title: NorcadiaCP_mcherry_R2877H_Ess6_RH-CA2-OLS_48

description:
REDI of CRISPRi gRNAs
processed data file: 1120_OLS45_48_all.analyzed.xlsx


title: DirMPCA-Mp3322_Sec14_RH-CA__2

status: Public on Feb 14 2017

type: SRA

city: Palo Alto

first: Robert

middle: Peter

state: CA

address: 3165 Porter Drive

department: Biochemistry

country: USA

institute: Stanford University

description:
Molecular probe library analysis
processed data file: Table_S2.xlsx

library_source: genomic

data_processing:
Library strategy: REDI
Sequence analysis of DNA library composition. Paired-end Illumina DNA sequencing was used to examine the composition of DNA libraries produced during this study. Amplification of the molecular probe library with DirMP-fwd and DirMP-rev primers produced amplicons of 276 bp in length. These amplicons were paired-end sequenced (2 x 250 cycles) to cover the entire length of the molecular probe sequences in both directions. To mitigate the confounding effect of sequencing-derived errors, analyses were restricted to sequences located between the molecular probe common amplification sites (TAGACGTAAGCCTGGTCTCA and ATCGGGAATCGAGTCTACCT) where the forward and reverse reads were in perfect agreement. In addition, the same sequencing run was used to analyze and compare the array- and yeast-derived libraries. The calculated sequencing error rates for this run (based on PhiX control DNA that was included in the sample) were 1.78% and 2.63% for read1 and read2, respectively. Based on these rates, sequencing artifacts are predicted to contribute ~0.01% of the observed errors (.0178 x 0.0263 x 0.25). Notably, an 8 nt random barcode was included in the design and synthesis of the molecular probes. These sequences become clonal after transformation into yeast and are no longer useful for improving quantification of target DNA (as they were originally intended). As the sequences of these barcodes are not known beforehand, they were excluded from the library composition analysis. Sequencing analysis of the molecular probe libraries revealed that 61.8% of the array-synthesized library and 3.6% of the library produced by REDI, did not match any of the probes that were targeted for selection (Figure 2B). We refer to these DNA sequences as ‘contaminating DNA’. These sequences were analyzed for similarity to a designed probe using bowtie2 (version 2.2.8) and samtools (version 1.2), and for hybrids (i.e. sequences where the first Homer is identical or similar to one probe, and where the second Homer is identical or similar to a different probe). For the library produced by REDI, we excluded sequencing reads that were associated with 66 clones that were selected (i.e. cherry-picked) but later determined to have a ‘petite’ phenotype (reduced growth due to spontaneous loss of mitochondrial DNA). We attempted to remove these clones by replica plating the entire collection onto growth medium that only supports respiration prior to creating the probe library. We still observed low levels of sequencing reads from these clones in the library. As they do not represent true contaminating DNA, they were excluded from the analysis presented in Figure 2.
Analysis of competitive growth assay data. Counts were generated by enumerating all sequences with perfect matches in both Read 1 and Read 2 of paired-end MiSeq data (i.e. requiring that the first 60 nucleotides of Read 1 containing the barcode and gRNA target complementarity were also present without mismatch in Read 2 (reverse complement)). These sequences were then mapped to the gRNAs that we had designed. Given the nature of our experimental design, we expect many strains to exhibit growth defects upon addition of ATc, and, consequently, produce a major change in the distribution of the resulting pool composition. This phenomenon created a unique challenge to the data normalization that is required when comparing across separate experiments. Widely used normalization methods, such as quantile normalization that assumes an unchanged shape of the distributions, or z-score normalization that assumes common mean (or median) across samples, cannot be applied.  To address this challenge, we developed a novel normalization strategy that relies on the identification of a set of “neutral” guides. Based on the experimental design, we make the following two assumptions: 1) many of the guides are damaging, i.e. lead to reduced fitness of the cells, and hence a reduced proportion of the guides in the pool; 2) a smaller but significant portion of the guides are neutral, i.e. they do not change the fitness of the cells, but their relative proportion will slightly increase in the pool due to the decreased fitness of the other strains. Based on these two assumptions, the following three steps were taken to define neutral guides:   A) We normalize all samples by the relative values of their respective total sequencing read counts. B)We clustered the log-transformed data using Euclidean distance measure and complete linkage algorithm, and chose a distance cutoff value of 11 to obtain the major clusters. C)We calculated the average log counts of all guides in each cluster and selected the cluster with the highest average counts as the neutral cluster. Guides in the neutral cluster had slightly increased counts in the ATc samples when compared to the non-ATc samples, consistent with the expected behavior of neutral guides.
Sequence analysis of diploid recombinants. Paired-end Illumina sequencing was used to identify the exogenous DNA physically linked to each barcode in the diploid recombinants. For each plate of recombinants, total genomic DNA was purified from ~150-200 million cells using the YeaStar Genomic DNA Kit (Zymo Research) and following the manufacturer’s directions. The genomic region of interest was PCR amplified from 1µl of purified genomic DNA template [98°C for 2 min, (98°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec) x 30 cycles, 72°C for 7 min], producing a 429 bp amplicon (molecular probe library) or a 941 bp amplicon (CRISPRi library). Typically, 240 cycles were used to sequence the exogenous DNA in the forward direction, and 60 sequencing cycles were used to sequence the barcode in the reverse direction. Briefly, sequencing reads were binned according to short (i.e. six nucleotide) indexing barcodes included in both the forward and reverse sequencing primers (index pairs were unique to each plate of diploid recombinants), and, then, by the 26 bp barcode (unique to each barcoder strain and, thus, colony position in the arrayed plate of recombinants). From each set of binned reads, the exogenous DNA sequence between the common priming regions was identified and compared to the designed molecular probe or guide RNA libraries. Sequences matching a designed sequence perfectly were of interest and were prioritized for selection. We further refined this list by de-prioritizing clones where the perfect match sequence was supported by fewer than 50 reads. In some cases, we also filtered diploid recombinants having multiple different sequences that perfectly matched multiple different designed sequences (likely indicating robotic picking of a ‘mixed colony’). Extending this last filter to accommodate partially matching sequences will mitigate contamination from mixed colonies (Figure S3) and will improve library quality.
Sequence analysis of probes missing or poorly represented from the molecular probe library. We identified 49 molecular probes that were absent or poorly represented in the molecular probe library following parsing in yeast. These probes were identified by fewer than 50 sequencing reads in the DNA library composition analyses. To explore explanations for the poor representation of these probes, we selected the 49 colonies representing these probes, collectively amplified the exogenous DNA (i.e. the amplified array-synthesized oligonucleotides that integrated into the target locus of the recipient strain), and sequenced those amplicons using MiSeq (Illumina). Sequences matching those found in the composition analyses of the original library were identified and are presented in different colors in Figure S3A. An examination of diploid recombinant sequences associated with the 49 indexing barcode pair/barcode combinations of the poorly represented clones are given in Figure S3B. Sequences represented by fewer than five reads were excluded from these analyses.
Molecular probe prepara

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: PCR of Custom Array Oligos

extract_protocol:
Total genomic DNA was purified from ~150-200 million cells using the YeaStar Genomic DNA Kit (Zymo Research) and following the manufacturer’s directions.
The genomic region of interest was PCR amplified from 1µl of purified genomic DNA template [98°C for 2 min, (98°C for 10 sec, 60°C for 20 sec, 72°C for 30 sec) x 30 cycles, 72°C for 7 min], producing a 429 bp amplicon (molecular probe library) or a 941 bp amplicon (CRISPRi library).

tag: sample number

tag: miseq run

tag: sample type


title: 902 (CLDP)

city: Tel Aviv

first: Metsada

last: Pasmanik-Chor

address: Ramat Aviv

department: Biology

country: Israel

institute: Tel Aviv University

library_source: transcriptomic

data_processing:
Data processing was performed using partek Flow build version 4.0.15.0702
Bases were trimmed on quality score=20. Adaptors were also trimmed.
Filter BAM files with minimal mapping quality=20.
Alignment usign Bowtie2 version 2.1.0.
Quantification to transcriptome performed using Partek E/M algorithm.
Genome_build: hg18
Supplementary_files_format_and_content: raw expression values

molecule: total RNA

organism: Homo sapiens

source_name: CLDP, ileal mucosa

growth_protocol: Biopsies were taken from the terminal ileum of the unoperated groups. Biopsies from pouch patients were taken by a single endoscopist (I.D.) from the most affected part within the pouch or from the middle of the pouch if there was no evidence of pathology.

extract_protocol:
Total RNA was extracted from frozen biopsies using Trizol® (Invitrogen, Carlsbad, CA) according to a standard protocol. Sample quality was assessed by both a photospectrometer (Nano Drop Technologies®, Thermo Scientific, Wilmington, DE) and agarose gels (1%). The 260/280 ratios in all samples were >1.8 and the 260/230 ratios were >1.7
Massive parallel (next-generation) sequencing (NGS) was carried out according to Illumina’s Small RNA sample preparation protocol (TruSeq Small RNA Sample Prep Kits, Illumina, San Diego, CA) for generating small RNA libraries directly from total RNA and for sequencing. miRNA libraries were sequenced on the HiSeq 2500 (Illumina) following the manufacturer’s protocol.

treatment_protocol: The biopsies were snap frozen and kept at -80ºC until RNA extraction. Scoring of histopathologic slides according to the histology sub-score of the PDAI was performed by a single IBD-trained pathologist (EB). Briefly, active inflammatory characteristics were scored according to neutrophil infiltration (0-3) and area of ulceration (0-3). Infectious or mechanical etiologies for pouch inflammation were excluded using microbiological stool assessment and endoscopy, respectively.

tag: disease state

value: CLDP

tag: tissue

value: ileal mucosa


title: NorcadiaCP_mcherry_R2877H_Ess6_RH-CA2-OLS_47

source_name: Yeast Genomic DNA


title: 1981 (NC)

source_name: NC, ileal mucosa

value: NC


title: NorcadiaCP_mcherry_R2877H_Ess6_RH-CA2-OLS_46


title: 52 (CLDP)


title: CA2-OLS_23-44_Norcadia-CA2-OLS_44

description:
REDI of CRISPRi gRNAs
processed data file: 1022_OLS23_44_all.analyzed.xlsx


title: NorcadiaCP_mcherry_R2877H_Ess6_RH-RH_101

description:
Molecular probe reaction with bacterial DNA
processed data file: Table_S4.xlsx


title: HKE3

city: Stockholm

first: Erik

last: Fasterius

address: AlbaNova University Center

department: Proteomics and nanotechnology

country: Sweden

institute: KTH Royal Institute of Technology

data_processing:
Alignment of reads using STAR (v2.5.1b)
Variant calling as per GATK's Best Practice workflow (v3.5.0, https://www.broadinstitute.org/gatk/guide/article?id=3891, December 2015)
Annotation using SnpEff/SnpSift (v4.2)
In-house Python and R scripts, as described in the corresponding article and R Markdown document.
Genome_build: GRCh37
Supplementary_files_format_and_content: .txt

source_name: colorectal cancer cell line HKE3

growth_protocol: Standard growth protocol in McCoy (HCT116, RKO) or DMEM media (HKE3), supplemented with 10 % FBS. Cells were split 1:5 every third or fourth day.

extract_protocol:
Rneasy Plus Mini Kit (Qiagen), as per manufacturer's instructions. All samples had a RIN value of 10, according to analysis on the Agilent 2100 BioAnalyzer.
TruSeq mRNA kit (Illumina), 200 ng per sample

treatment_protocol: No treatments.

tag: cell line

tag: cell type

value: Colorectal carcinoma-derived cells


title: RKO

source_name: colorectal cancer cell line RKO

value: RKO


title: CA2-OLS_23-44_Norcadia-CA2-OLS_42


title: Cond.R2877H_MP-49mol_Sec14_RH_CANIY-MP_49mol

description:
Molecular probe library analysis
processed data file: Table_S5.xlsx


title: NorcadiaCP_mcherry_R2877H_Ess6_RH-CA2-OLS_45


title: CA2-OLS_23-44_Norcadia-CA2-OLS_43


title: 1632 (NC)


title: 1471 (NC)


title: HCT116

source_name: colorectal cancer cell line HCT116


title: CA2-OLS_23-44_Norcadia-CA2-OLS_40


title: CA2-OLS_23-44_Norcadia-CA2-OLS_38


title: HTR siH19 rep5

city: Farmington

first: Tingting

last: Geng

state: CT

address: 263 Farmington Avenue

department: Department of Immunology

institute: Uconn Health

data_processing:
RTA 2.1.3 software was used for basecalling.
Alignments were made using Tophat 2.0.7, FPKM estimation of reference genes and transcripts were made using Cufflinks2 (TopHat Alignment 1.0.0 embedded in Galaxy ).
Relative expression of genes and transcripts between siCon and siH19 samples was determined using Cuffdiff 2.1.1.
Genes and transcripts that have significant differentiatial expression between siCon and siH19 triplicates (q Value <0.05) were presented in the processed data file.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data file includes averaged FPKM values for each condition.

source_name: trophoblast_siH19 (H19 Knockdown)

growth_protocol: RPMI-1640 medium, supplemented with 10% fetal bovine serum, heat inactivated, 1% penicillin/streptomycin, 1% L-glutamine

extract_protocol:
Total RNAs were extracted from cells using PureLink RNA Mini Kit.
RNA transcripts are enriched by selectively depleting ribosomal RNA molecules using the Ribo-Zero Gold Kit (Epicentre) and then sheared by incubation at 94C°. Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries. The cDNA library is then endrepaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Perkin Elmer LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.

treatment_protocol: Cells were transfected in a 48-well plate scale. Cells were transfected with either control siRNA or H19 siRNA. After 48h later, cells were harvested for RNA extraction.

tag: transfection

tag: genotype/variation

value: H19 knockdown

tag: time

value: 48h after transfection


title: HTR siH19 rep6


title: HTR siH19 rep4


title: CA2-OLS_23-44_Norcadia-CA2-OLS_41


title: CA2-OLS_23-44_Norcadia-CA2-OLS_37


title: HTR siH19 rep3


title: HTR siH19 rep2


title: 382 (CLDP)


title: HTR siCon rep6

source_name: trophoblast_Control siRNA

value: siControl

value: control


title: CA2-OLS_23-44_Norcadia-CA2-OLS_34


title: CA2-OLS_23-44_Norcadia-CA2-OLS_35


title: CA2-OLS_23-44_Norcadia-CA2-OLS_36


title: HTR siH19 rep1


title: HTR siCon rep5


title: CA2-OLS_23-44_Norcadia-CA2-OLS_33


title: LJA289

city: Wuhan

first: Yang

last: Zhou

state: Hubei

address: Shizishanjie #1

department: College of animal science

country: China

institute: Huazhong Agricultural University

data_processing: All the RNA-seq data were filtered for removing the adapter sequences, contamination and low-quality reads, and the clean reads were aligned to the modified cattle reference genome (UMD3.1). We applied Tophat (Version 2.0.13) and Cufflink (Version 2.2.1) protocols according to the previously published paper using the default parameters

organism: Bos taurus

source_name: Cattle

extract_protocol:
All samples were stored at -80°C until sonicated or pulverized and then homogenized using a polytron homogenizer for extraction of total RNA with TRIZOL (Molecular Research Center, Cincinnati, OH, USA), using the manufacturer's protocol.
The libraries were constructed according to the TruSeq RNA Library Prep Kit v2 standard protocol

value: nucleated blood cells


title: LJA285

value: rumen


title: LJA281

value: ileum


title: CA2-OLS_23-44_Norcadia-CA2-OLS_32


title: HTR siCon rep3


title: CA2-OLS_23-44_Norcadia-CA2-OLS_39


title: HTR siCon rep4


title: LJA274

value: abomasum


title: HTR siCon rep1


title: HTR siCon rep2


title: CA2-OLS_23-44_Norcadia-CA2-OLS_30


title: LJA263

value: mammary/parenchyma


title: CA2-OLS_23-44_Norcadia-CA2-OLS_29


title: COR-B

city: Danzhou

first: Wu

last: Shaohua

state: Hainan

address: Baodao Xincun

department: Rubber Research Institute

description:
one year old Hevea epicormic shoots
the mixed RNA samples of 8 h, 1 d, 2 d and 3 d after coronatine treatment

data_processing:
Filter Dirty Raw Reads: Raw reads that only have 3' adaptor fragments should be removed before data analysis
Clean Reads:Reads after filtering dirty raw reads, on which all following analysis are based
Assembly:Transcriptome de novo assembly is carried out with short reads assembling program - SOAPdenovo (Li, Zhu et al. 2009). SOAPdenovo firstly combines reads with certain length of overlap to form longer fragments without N, which are called contigs. Then the reads are mapped back to contigs; with paired-end reads it is able to detect contigs from the same transcript as well as the distances between these contigs. Next, SOAPdenovo connects the contigs using N to represent unknown sequences between each two contigs, and then Scaffolds are made. Paired-end reads are used again for gap filling of scaffolds to get sequences with least Ns and cannot be extended on either end. Such sequences are defined as Unigenes. When multiple samples from a same species are sequenced, Unigenes from each sample's assembly can be taken into further process of sequence splicing and redundancy removing with sequence clustering software to acquire non-redundant Unigenes as long as possible. In the final step, blastx alignment (evalue < 0.00001) between Unigenes and protein databases like nr, Swiss-Prot, KEGG and COG is performed, and the best aligning results are used to decide sequence direction of Unigenes. If results of different databases conflict with each other, a priority order of nr, Swiss-Prot, KEGG and COG should be followed when deciding sequence direction of Unigenes. When a Unigene happens to be unaligned to non of the above databases, a software named ESTScan (Iseli, Jongeneel et al. 1999) will be introduced to predict its coding regions as well as to decide its sequence direction. For Unigenes with sequence directions, we provide their sequences from 5' end to 3' end; for those without any direction we provide their sequences from assembly software.
Genome_build: All-Unigene.fa
Supplementary_files_format_and_content: [.txt] FPKMs and differentially expressed unigenes between two clones

organism: Hevea brasiliensis

source_name: COR, late

growth_protocol: H. brasiliensis clones CATAS7-33-97 grown at the Experimental Station of the Rubber Research Institute of the Chinese Academy of Tropical Agricultural Sciences

extract_protocol:
Total RNA was isolated from bark tissues according to the procedures of Tian et al.(2013). RNA integrity was confirmed using the 2100 Bioanalyzer (Agilent Technologies). Beads with Oligo(dT) were used to isolate poly(A) mRNA after total RNA (QIAGEN GmbH, Hilden, Germany). Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature.
Taking these short fragments as templates, the first-strand cDNA was synthesized by using random hexamer-primer and Superscrip (Invitrogen California, USA). The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit (QIAGEN GmbH, Hilden, Germany) and resolved with EB buffer for end reparation and adding poly(A). Then the short fragments were connected with sequencing adapters. After agarose gel electrophoresis, the suitable fragment was selected for the PCR amplification as templates.

treatment_protocol: The epidermis and part of the cortex within area of 2 cm × 4 cm at the middle of the EU2 stem were removed by scratching with a single side razor blade. The wounded surface was immediately wrapped with a polyethylene membrane after being respectively applied with 20 μM coronatine (Sigma, USA) and sterile water.

tag: clone

tag: agent

value: coronatine

value: 8 h, 1 d, 2 d and 3 d

value: bark


title: COR-A

institute: Chinese Academy of Tropical Agricultural Sciences

description:
one year old Hevea epicormic shoots
the mixed RNA samples of 1 h, 2 h and 4 h after coronatine treatment

source_name: COR, early

value: 1 h, 2 h and 4 h


title: LJA249

value: uterus intercaruncular


title: CA2-OLS_23-44_Norcadia-CA2-OLS_28


title: LJA219

value: frontal cortex


title: LJA167

value: whole testes


title: Control-B

description:
one year old Hevea epicormic shoots
the mixed RNA samples of 8 h, 1 d, 2 d and 3 d after water treatment

source_name: Control, late


title: CA2-OLS_23-44_Norcadia-CA2-OLS_27


title: Control-A

description:
one year old Hevea epicormic shoots
the mixed RNA samples of 1 h, 2 h and 4 h after water treatment

source_name: Control, early


title: CA2-OLS_23-44_Norcadia-CA2-OLS_31


title: CA2-OLS_23-44_Norcadia-CA2-OLS_23


title: CA2-OLS_23-44_Norcadia-CA2-OLS_25


title: BGA60

data_processing:
After output data, the raw reads were filtered for removing the adapter sequences, contamination and low-quality reads. The clean reads were then aligned to the modified cattle reference genome (UMD3.1) by SOAPaligner. Every hit with a single placement with minimum number of mismatches and a clear strand assignment was defined as an unambiguous alignment and was used in methylcytosine ascertainment.
Genome_build: UMD3.1

source_name: abomasum

extract_protocol:
Genomic DNA for each tissue was isolated according to the protocol of QIAamp@ DNA Mini Kit (QIAGEN).
RRBS libraries were constructed according to the manufacturer’s instruction. In detail, 3ug genomic DNA was digested with the methyl insensitive MspI enzyme (CCGG site) at 37 ºC for 16h for each samples, individually. The digested DNA products were purified using QIAquick PCR Purification Kit (QIAGEN) and added single A nucleotides in the blunt-end, which were then ligated to methylated adapter with T overhang. The DNA fragments with 150-400bp length were collected and purified using 2.5% agarose gel electrophoresis. The recovered DNA was treated with methylation-Gold Kit (ZYMO) to apply for the bisulfite conversion. The bisulfite-converted DNA was finally amplified by PCR to construct the RRBS libraries. The Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and the real-time quantitative PCR (qPCR, TaqMan Probe) were used to quantitate and quantify the RRBS libraries, separately. The qualified libraries were amplified on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3-cBot-HS, Illumina).


title: BGA135

source_name: nucleated blood cells


title: BGA173

source_name: 90 d lactating gland

value: 90 d lactating gland


title: BGA62

source_name: ileum


title: CA2_OLS1to22_mCherry-CA2-OLS_8

description:
REDI of CRISPRi gRNAs
processed data file: 1005_OLS6_22_all.analyzed.xlsx


title: CA2_OLS1to22_mCherry-CA2-OLS_7


title: CA2-OLS_23-44_Norcadia-CA2-OLS_24


title: CA2_OLS1to22_mCherry-CA2-OLS_9


title: CA2_OLS1to22_mCherry-CA2-OLS_6


title: BGA81

source_name: rumen


title: BGA22

source_name: uterus intercaruncular


title: CA2_OLS1to22_mCherry-CA2-OLS_22

source_name: Yeast Genomic DNA


title: BGA47

source_name: frontal cortex


title: CA2_OLS1to22_mCherry-CA2-OLS_21


title: BGA19

source_name: mammary/parenchyma


title: CA2-OLS_23-44_Norcadia-CA2-OLS_26


title: CA2_OLS1to22_mCherry-CA2-OLS_20


title: CA2_OLS1to22_mCherry-CA2-OLS_18


title: BGA14

source_name: whole testes


title: BGA13

source_name: skeletal muscle near ceasarian

value: skeletal muscle near ceasarian


title: CA2_OLS1to22_mCherry-CA2-OLS_16


title: CA2_OLS1to22_mCherry-CA2-OLS_19


title: CA2_OLS1to22_mCherry-CA2-OLS_14


title: CA2_OLS1to22_mCherry-CA2-OLS_15


title: YFP_MI_SCApYFPp4

city: Madrid

first: Antonio

last: Bernad

address: Melchor Fernandez Almagro, 3

country: Spain

institute: Spanish Cardiovascular Research Center

data_processing:
Libraries were applied to an Illumina flow cell for cluster generation (True Seq SR Cluster Kit V2 cBot). Single reads of 75 bases were generated on the Genome Analyzer IIx using the TruSeq SBS Kit v5 (Illumina) and following the standard sequencing protocol.
Reads were pre-processed with Cutadapt  to remove TruSeq adapter sequences and OVATION primer sequences.
Resulting reads were mapped on the mouse transcriptome (Ensembl gene-build NCBIM37.v65) using RSEM.
Data were normalized and differential expression tested using the Bioconductor package EdgeR.
Genome_build: NCBIM37.v65.
Supplementary_files_format_and_content: matrix_table_homeostasis.xls file contains ENSEMBL gene IDs, MGI gene names, normalized counts per million for each sample, fold change, log fold change and adjusted p_value.
Supplementary_files_format_and_content: matrix_table_MI.xls file contains ENSEMBL gene IDs, MGI gene names, normalized counts per million for each sample, fold change, log fold change and adjusted p_value.

organism: Mus musculus

source_name: Bmi1+ cardiac stem cells MI

extract_protocol:
Hearts were perfused with PBS to remove blood cells and processed by enzymatic digestion using 0.1% collagenase IV (Sigma) and 10 μg/ml DNAse (Roche) for 40 minutes at 37ºC. Samples were then mechanically disrupted  and filtered. Flow cytometry staining was performed to isolate Sca-1+Bmi1-cells and Bmi1+ cells from adult hearts. Bmi1+ cells were isolated due to YFP detection.Isolated Sca-1+ Bmi1-(YFP-) CD45- and Bmi1+ (YFP+) were isolated by sorting. In the homeostasis RNAseq, 4 replicates were performed (n=6 animals each replicate). In the MI RNAseq 4 replicates were performed (n=2-3 animals each replicate).
Total RNA was isolated with the Arcturus PicoPure kit (Applied Biosystems, Carlsbad, CA, USA). cDNA was amplified from total RNA (10-25ng) using the Ovation® RNA-seq System v2 (NuGEN® Technologies, San Carlos CA).
Amplified cDNA (1μg) was sonicated to an average size of 200-300 bp and used with the TruSeq DNA Sample Preparation v2 Kit (Illumina, San Diego CA) to generate index-tagged sequencing libraries.

treatment_protocol: Bmi1IresCreER mice were induced with tamoxifen (9 mg per 40 g of body weight) every 24 h on three consecutive days. After 5 days the hearts were harvested for homeostatic condition. For myocardial infarction,  animals were induced with tamoxifen and 5 days later underwent MI. 5 days after the infarction, the hearts were harvested.

value: Bmi1+ cardiac stem cells

value: MI

tag: strain

value: heart

tag: developmental stage

value: adult


title: YFP_MI_SCApYFPp3


title: SCA_WT_SCApYFPn4

source_name: Sca-1+Bmi1- cardiac progenitor cells homeostasis

value: Sca-1+Bmi1- cardiac progenitor cells

value: homeostasis


title: SCA_WT_SCApYFPn3


title: YFP_MI_SCApYFPp2


title: YFP_MI_SCApYFPp1


title: CA2_OLS1to22_mCherry-CA2-OLS_13


title: SCA_WT_SCApYFPn1


title: YFP_WT_SCApYFPp4

description:
matrix_table_homeostasis.xls
matrix_table_MI.xls

source_name: Bmi1+ cardiac stem cells homeostasis


title: YFP_WT_SCApYFPp3


title: YFP_WT_SCApYFPp2


title: OLS63-79_ERG1-4_2877.001H-OLS_79

description:
REDI of molecular probes
processed data file: 0504_all.analyzed.OLS63-79.xlsx


title: CA2_OLS1to22_mCherry-CA2-OLS_10


title: CA2_OLS1to22_mCherry-CA2-OLS_11


title: YFP_WT_SCApYFPp1


title: OLS63-79_ERG1-4_2877.001H-OLS_76


title: OLS63-79_ERG1-4_2877.001H-OLS_77


title: Lox replicate 4

city: Sydney

first: Peter

last: White

address: High Street

country: Australia

institute: University of New South Wales

description: cuffdiff mock vs lox.txt

data_processing:
Sequencing reads were assesed for quality and base-calling using FastQC (v.0.10.1)
Poor quality bases were trimmed from reads using Trimmomatic (v0.32.2)
Reads mapped to mm10 genome using TopHat (v0.9) with default parameters
Transcripts were assembled using Cufflinks (v2.2.1.0) and subsequentl differentialy analysis between time points was perfored using Cuffdiff (v2.2.1.2)
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text file of Cuffdiff output

source_name: Macrophage cell line

growth_protocol: RAW264.7 cells were grown in standard DMEM supplemented with FBS, Glutamax and Pen/Strep and passaged as per standard tissue culture protocols.

extract_protocol:
Cells were harvested from monolayers and growth media by centrifugation and total RNA was extracted using TRIzol (Invitrogen). RNA was further cleaned using the Rneasy kit (Qiagen) with DNase treatment.
RNA samples were ribodepleted using RiboZero Gold and libraries prepared using Ilumina TruSeq Stranded Total RNAseq kit.

treatment_protocol: 1x10^7 cells were treated with mock treated with growth media or infected with MNV at an MOI of 5 for 4, 8, 12, 16 and 20 hrs.

tag: infection

value: Lox


title: CA2_OLS1to22_mCherry-CA2-OLS_17


title: OLS63-79_ERG1-4_2877.001H-OLS_78


title: TBP

city: Boston

first: Yoo Jin

last: Joo

state: MA

address: 240 Longwwod Ave.

department: BCMP

institute: HMS/Harvard University

description: MATα, ura3-, leu2-3,112, trp1-1, his3-11,15, fpr1::NAT, RPL13A-2xFKBP12::TRP1, tor1-1, ade2-1, can1-100

data_processing:
Illumina Casava software used for basecalling.
Sequenced reads mapped to sacCer3 whole genome using bowtie v1.1.1 with parameters -S -p 4 -m 1
Peaks were called using Macs2 (v2.1.0) with parameters –c -f BAM -g 12100000 -B -q 0.01 --nomodel --extsize 150 --SPMR
Genome_build: sacCer3 (R64)
Supplementary_files_format_and_content: BedGraph files generated by Macs2 possessing SPMR value.

source_name: YF2229, TBP ChIP

growth_protocol: Yeast cells were grown in rich media (YPD) and harvested at early exponential growing stage (OD600 was not over 0.5) after crosslinking with 1% formaldehyde.

extract_protocol:
Sheared chromatin (100 bp ~ 300 bp) was prepared using a Misonix sonicator 300 with cup horns. Crosslinked chromatin was precipitated by standard methods with specific antibodies.
Libraries were prepared according to previous methods (Wong, K.H., Jin, Y., and Moqtaderi, Z. (2013). Multiplex Illumina sequencing using DNA barcoding. Curr Protoc Mol Biol Chapter 7, unit7.11.-7.11.11).

tag: genotype

tag: Stage

value: early exponential growing

tag: chip antibody

value: anti-TBP rabbit polyclonal (custom made)


title: Lox replicate 2

description: cuffdiff mock vs lox.txt


title: CA2_OLS1to22_mCherry-CA2-OLS_12

tag: sample number

tag: miseq run


title: SCA_WT_SCApYFPn2

tag: disease state


title: H4ac

description: MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0

source_name: BY4741, H4ac ChIP

value: MATa, ura3delta0, leu2delta0, his3delta1, met15delta0

value: anti-acetyl-histone H4 (Millipore, 06-598)


title: Lox replicate 1

description: cuffdiff mock vs lox.txt


title: Input DNA

description: MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0

source_name: BY4741, input


title: H3

description: MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0

source_name: BY4741, H3 ChIP

value: anti-histone H3 (abcam, ab1791)


title: OLS63-79_ERG1-4_2877.001H-OLS_72

tag: sample type


title: OLS63-79_ERG1-4_2877.001H-OLS_71


title: OLS63-79_ERG1-4_2877.001H-OLS_73


title: MNV replicate 3

description: cuffdiff mock vs mnv.txt

tag: cell line

value: MNV


title: OLS63-79_ERG1-4_2877.001H-OLS_70


title: MNV replicate 2

description: cuffdiff mock vs mnv.txt


title: OLS63-79_ERG1-4_2877.001H-OLS_69


title: MNV replicate 1

description: cuffdiff mock vs mnv.txt


title: Mock replicate 4

value: Mock


title: Mock replicate 3


title: Lox replicate 3

description: cuffdiff mock vs lox.txt


title: OLS63-79_ERG1-4_2877.001H-OLS_75


title: Mock replicate 2


title: DirMPCA-Mp3322_Sec14_RH-MP__3

description:
Molecular probe library analysis
processed data file: Table_S3.xlsx


title: Mock replicate 1


title: OLS63-79_ERG1-4_2877.001H-OLS_67


title: OLS63-79_ERG1-4_2877.001H-OLS_66


title: OLS63-79_ERG1-4_2877.001H-OLS_65


title: OLS63-79_ERG1-4_2877.001H-OLS_74


title: MNV 20 hpi

tag: time point

value: 20 hpi


title: Cherry_Pick_Sewage_RAS_Sec14_OLS58-62_tiling-OLS62

description:
REDI of molecular probes
processed data file: 0423_all.analyzed.OLS58-62.xlsx


title: OLS63-79_ERG1-4_2877.001H-OLS_63


title: MNV 16 hpi

value: 16 hpi


title: MNV replicate 4

description: cuffdiff mock vs mnv.txt


title: Cherry_Pick_Sewage_RAS_Sec14_OLS58-62_tiling-OLS61


title: Cherry_Pick_Sewage_RAS_Sec14_OLS58-62_tiling-OLS60


title: MNV 4 hpi

value: 4 hpi


title: MNV 8 hpi

value: 8 hpi


title: 32 Weeks Whole Lung Control

city: Rochester

first: Tyler

last: Beach

state: NY

address: 601 Elmwood Ave., Box 850

institute: University of Rochester

data_processing:
DATA FORMATTING (Illumina -> FastQ) & DEMULTIPLEXING - bcltofastq-1.8.4
CLEANING - Trimmomatic-0.32 "SLIDINGWINDOW:4:20 TRAILING:13 LEADING:13 ILLUMINACLIP:adapters.fasta:2:30:10 MINLEN:15"
MAPPING - STAR_2.4.2a "--runMode alignReads --genomeDir ${GENOME} --readFilesIn ${SAMPLE} --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical"
DIFFERENTIAL EXP. - cufflinks-2.0.2 (cuffdiff) "--library-type fr-firststrand --FDR 0.05 -u -b GENOME"
Genome_build: GRCm38.p4 + Gencode-M6 Annotation
Supplementary_files_format_and_content: tab-delimited text files include raw or FPKM values for each Sample

source_name: Whole lung cell suspension

extract_protocol:
Qiagen RNeasy PLUS.  RNA from 3 mice per pool.
Clontech ultralow, Illumina NexteraXT

treatment_protocol: 12.5 Gy Whole Thorax γ-irradiation, 0 Gy age matched controls

tag: timepoint

tag: treatment

value: Control

tag: cell type

value: Whole lung cell suspension


title: Sewage_RAS_Sec14_OLS-OLS45

description:
REDI of molecular probes
processed data file: 0402_all.analyzed.OLS39-45.xlsx


title: Cherry_Pick_Sewage_RAS_Sec14_OLS58-62_tiling-OLS58


title: Cherry_Pick_Sewage_RAS_Sec14_OLS58-62_tiling-OLS59


title: 4 Weeks Whole Lung Radiated

value: Radiated


title: 12 Weeks Whole Lung Radiated


title: 12 Weeks Whole Lung Control


title: Sewage_RAS_Sec14_OLS-OLS43


title: 32 Weeks Whole Lung Radiated


title: 1 Week Whole Lung Radiated


title: Sewage_RAS_Sec14_OLS-OLS44


title: 4 Weeks Whole Lung Control


title: 24 Hrs Whole Lung (WL) Radiated


title: Sewage_RAS_Sec14_OLS-OLS41


title: Sewage_RAS_Sec14_OLS-OLS42


title: Sewage_RAS_Sec14_OLS-OLS39


title: 1 Week Whole Lung Control


title: Sewage_RAS_Sec14_OLS-OLS40


title: Mock


title: 24 Hrs Whole Lung (WL) Control


title: OLS63-79_ERG1-4_2877.001H-OLS_64


title: IU6173_RNA-seq rep 3

city: Bloomington

first: Malcolm

middle: E

last: Winkler

state: IN

address: 1001 E. Third Street

institute: Indiana University

description: ∆phoU2::kanrpsL+ ∆phoU1::Pcerm

data_processing:
Bases were called using bcl2fasta v2.16.0.10
Reads were quality and adapter trimmed using Trimmomatic v0.33 using default parameters, Illumina adapters, and a minimum read length of 35bps
Mapping was accomplished with bowtie2 version 2.1.0 by mapping to chromosome NC_008533 and the plasmid NC_005022
Differential gene expression was identified using the program DESeq with these non-default parameters: estimateDispersions was set to "blind" and the sharingMode as "fit-only"
Genome_build: NC_008533.1
Supplementary_files_format_and_content: raw counts of sequencing reads for the features of interest including genes and intergenic regions for each fastq file; intergenic regions are identified as follows Inter.<molecule>.<strand>.<basepair_begin>.<basepair_end>

organism: Streptococcus pneumoniae

source_name: Exponentially grown cells

growth_protocol: Cultures of strains IU3116 (cep::kanrpsL+), IU6139 (∆phoU2::kanrpsL+), and IU6173 (∆phoU2::kanrpsL+ ∆phoU1::Pcerm) grown exponentially in BHI media at 37ºC to OD620 ≈0.15 to 0.2. Starter cultures were used to inoculate 30 mL cultures of BHI broth in 50 mL conical tubes.

extract_protocol:
To isolate RNA for RNA-sequencing strains were grown in 30 ml of BHI starting at an OD620 of 0.002 in 50 ml conicals. RNA was extracted from exponentially growing cells (OD620 ~0.15) using the FastRNA Pro Blue Kit (MP Bio) according to the manufacturer’s guidelines. Briefly, cells were collected by centrifugation at 14,500 x g for 5 minutes at 4oC.  Cell pellets were resuspended in 1 ml of RNAproTM solution (MP Bio) and processed three times in the Fast Prep Instrument (MP Bio) for 40 seconds at a speed setting of 6.0. Chloroform and 100% ethanol was used to extract and precipitate RNA from the lysate. RNA purification was done using miRNeasy minikit (Qiagen), including on-column treatment with DNase I (Qiagen), following the manufacture’s guidelines. The amount and purity of all RNA samples isolated were assessed by NanoDrop spectroscopy (Thermo Fisher). RNA integrity of the samples used for RNA-seq library preparation was further assessed using the Agilent 2100 BioAnalyzer (Aligent Technologies).
cDNA libraries were prepared from total RNA by the University of Wisconsin-Madison Biotechnology Center. mRNA was enriched from two micrograms total RNA using RiboZeroTM rRNA Removal (Gram-positive bacteria) Kit (EpiCentre Inc., Madison, WI, USA). rRNA-depleted mRNA samples were purified by ethanol precipitation and quantified by fluorometry with the Qubit® RNA assay kit (Invitrogen, Carlsbad, CA, USA).Double stranded cDNA synthesis was performed following  ScriptSeqTM v2 RNA-Seq Library Preparation guide (EpiCentre Inc., Madison, WI, USA) in accordance with the manufacturer’s standard protocol. Thirty nanograms of enriched mRNA was fragmented using divalent cations via incubation for 5 min at 85oC. The first strand of cDNA was synthesized by reverse transcription using random-sequence primers containing a tagging sequence at their 5’ ends. Di-tagged cDNA was synthesized by random annealing of a terminal-Tagging Oligo (TTO) to the 3’ end of the cDNA for extension of the cDNA by DNA polymerase. Di-tagged cDNA was purified using Agencourt AMPure® XP beads (Beckman Coulter, Indianapolis IN, USA) followed by PCR amplification for 15 cycles using FailsafeTM PCR enzyme and ScriptSeq Index DNA primer set (EpiCentre Inc., Madison, WI). This step generated the second strand of cDNA and completed the addition of Illumina adapter sequences incorporating a user-defined barcode. The amplified libraries were purified using Agencourt AMPure® XP beads. Quality and quantity were assessed using an Agilent DNA 1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA) and Qubit® dsDNA HS assay kit (Invitrogen, Carlsbad, California, USA), respectively. Libraries were standardized to 2 μM. Cluster generation was performed using standard Cluster kits (v3) and Illumina Cluster Station.
Single-end 100bp sequencing was performed using standard SBS chemistry (v3) on an Illumina HiSeq2000 sequencer. Images were analyzed using the standard Illumina pipeline, version 1.8.2.

tag: mutation


title: IU6173_RNA-seq rep 2

description: ∆phoU2::kanrpsL+ ∆phoU1::Pcerm


title: MNV 12 hpi

value: 12 hpi


title: PFC_WT_3

first: Ryan

last: Walsh

state: Massachusetts

address: 185 Cambridge St. CPZN 4100

institute: Harvard

data_processing:
Basecalls performed using CASAVA version 1.7
Subsequent to demultiplexing and adapter trimming, fastqs were mapped to mm10 with splice-aware aligner STAR vers-2.3.0 using mm10 Ensembl gtf
To calculate RPKM expression values, sequencing reads were counted over an assembly of all putative exons for each gene and normalized by the length of the exon assembly and by the size of the library.
Genome_build: mm10
Supplementary_files_format_and_content: Gene counts for each sample are given in tab-delimited txt files.

growth_protocol: ESCs were derived from blastocyst outgrowths and maintained on gelatin/feeders. NPCs were derived from E16.5 embryos and maintained in monolayer on polyornitine.

extract_protocol:
mRNA was harvested from cells and tissues using Qiazol and cleaned up with an mRNAeasy kit.
Libraries were constructed with the Illumina TruSeq RNA prep kit following the manufacturer's protocol

tag: strain background

tag: genotype/variation

value: wild type

tag: age

tag: tissue/cell type

value: Prefrontal Cortex


title: PFC_KO_2

value: Phf8 KO


title: Glycine2

city: La Jolla

first: Bernhard

last: Palsson

address: 9500 Gilman Dr.

department: Bioengineering

institute: UCSD

description: 10 mM Glycine in Media

data_processing:
Sequenced reads were mapped onto NC_000913 reference genome sequence using bowtie v0.12.8 with parameters -X 1000 -n 2
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using cufflinks v2.1.1 with the option --library-type fr-firststrand
Genome_build: ASM584v2
Supplementary_files_format_and_content: comma-delimited text files include RPKM values for each Sample.

organism: Escherichia coli BW25113

source_name: Total RNA isolated from E. coli

growth_protocol: Cells were grown to mid-log phase aerobically at 37°C in M9 Minimal Media 4 g/L D-Glucose with 10 mM of the corresponding amino acid supplementation

extract_protocol:
Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen Inc., Valencia, CA, USA) and genomic DNA was removed by gDNA Eliminator spin column in the RNeasy Plus Mini Kit. RNA quality and concentration was determined by analysis with a NanoDrop 1000 (Thermo Scientific Inc., Wilmington, DE, USA).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The cell culture was treated with the RNAprotect reagent (Qiagen).

tag: strain

tag: genotype

value: WT

tag: amino acid supplementation

value: 10 mM Glycine in Media


title: Threonine1

description: 10 mM L-Threonine in Media

value: 10 mM L-Threonine in Media


title: Threonine2

description: 10 mM L-Threonine in Media


title: PFC_KO_1


title: Pyruvate 2

description: 3.3 g/L Sodium Pyruvate

growth_protocol: Cells were grown to mid-log phase aerobically at 37°C with a carbon-source supplemented minimal media (211 mM Na2HPO4, 110 mM KH2PO4, 42.8 mM NaCl, 56.7 mM (NH4)2SO4, 6.3 uM ZnSO4, 7uM CuCL2, 7.1 uM MnSO4, 7.6 uM CoCl2, 0.1 mM CaCl2, 1 mM MgSO4, 1.4 mM thiamine, 60 uM FeCl3 at pH 7)

tag: carbon source

value: 3.3 g/L Sodium Pyruvate


title: Glycine1

description: 10 mM Glycine in Media


title: PFC_WT_1


title: PFC_WT_2


title: IU3116_RNA-seq rep 1


title: IU3116_RNA-seq rep 3


title: IU3116_RNA-seq rep 2


title: IU6139_RNA-seq rep 1


title: IU6139_RNA-seq rep 3


title: IU6173_RNA-seq rep 1

description: ∆phoU2::kanrpsL+ ∆phoU1::Pcerm


title: IU6139_RNA-seq rep 2


title: Pyruvate 1

description: 3.3 g/L Sodium Pyruvate


title: PFC_KO_3


title: STR_KO_3

value: Ventral Striatum


title: STR_KO_2


title: STR_KO_1


title: OLS63-79_ERG1-4_2877.001H-OLS_68

tag: sample number

tag: miseq run


title: Glucose 2

description: 5.0 g/L D-Glucose in Media

value: 5.0 g/L D-Glucose in Media


title: STR_WT_3


title: STR_WT_2


title: STR_WT_1


title: NPC_KO_3

value: Neural Progenitor Cells (NPC)


title: NPC_KO_2


title: NPC_WT_2


title: exploratory replicate 1

city: Hamilton

first: stephanie

middle: erin

last: jones

state: ON

address: 1280 Main St W

country: Canada

institute: McMaster University

data_processing:
Fastq files from the same sample were concatenated to produce one fastq file (shown as raw files)
Reads were aligned to the S. venezuelae genome using Bowtie2, then sorted, indexed, and converted to BAM format using SAMtools for visualization in Integrated Genomics Viewer
Raw transcript levels, normalized transcript levels, RPKM transcript levels, and expression transcript levels (transcript levels normalized by the upper quartile of gene expression) were obtained using Rockhopper software (McClure et al., 2013)
For Rockhopper, raw concatenated fastq files from Illumina were used as input. Default parameters were used, with reverse complementation of reads
Supplementary_files_format_and_content: abundance measurements (normalized transcript levels)

organism: Streptomyces venezuelae ATCC 10712

source_name: S. venezuelae cells

growth_protocol: S. venezuelae explorer cells were grown beside the yeast S. cerevisiae on YPD agar plates (yeast extract-peptone-glucose) for 14 days at 30ºC, and S. venezuelae static cells were grown alone on YPD agar for 24 hours.

extract_protocol:
RNA was isolated using guanidium thiocyanate, followed by acid phenol-chloroform extractions and DNase treatments
rRNA was depleted using a ribo-zero rRNA depletion kit. RNA libraries were prepared for sequencing using standard Illumina protocols.

value: 14-day old explorer cells


title: ESC_KO_2

value: Embryonic Stem Cells (ESC)


title: NPC_KO_1


title: NPC_WT_3


title: Acetate 1

description: 3.5 g/L Sodium Acetate in Media

value: 3.5 g/L Sodium Acetate in Media


title: exploratory replicate 2


title: Fumarate 1

description: 2.8 g/L Disodium Fumarate

value: 2.8 g/L Disodium Fumarate


title: Fumarate 2

description: 2.8 g/L Disodium Fumarate


title: static replicate 2

value: 1-day old static cells


title: Glucose 1

description: 5.0 g/L D-Glucose in Media


title: static replicate 1


title: Acetate 2

description: 3.5 g/L Sodium Acetate in Media


title: NPC_WT_1


title: Morus alba L. drought stressed mulberry degradome

city: zhenjiang

first: li

middle: rui

last: xue

state: jiangsu

address: sibaidu

department: School of Biology and Technology

institute: Jiangsu University of Science and Technology

data_processing:
Library strategy: degradome-seq
By using Illumina Hiseq 2000 sequencing system, degradome sequencing takes SE51 sequencing strategy and produces 50nt raw reads.Data preprocessing using Perl script, and the script includes the following functions:1. Remove the reads with adapter; 2. Remove the low quality reads; 3. Remove the reads with N; 4. Summarize the unique tags and total tags.
After preprocssing, clean tags are generated and stored.The type of degradome tags (unique tag) and the quantity of degradome tags (total tag) will be counted.
Map clean tags to genome by SOAP2.20 with parameters -M 0 -v 2 -r 2 and analyze the genome distribution of tags.
Identify and get rid of ncRNA by the alignment to Rfam and Genbank databases, and annotate the cleaved genes by searching kegg and nr databases using BLAST with parameters -F F -e 0.01 .
Identification of miRNAs degradation site and the miRNA sequences used in this analysis from mirbase21.0. There are 5 steps in this analysis process: Step 1, mapping tags to reference genes using SOAP2.20 and the cDNA sequences can be used as reference genes. Step 2, summarize the information of cleaved sites and define categories (density file) using CleaveLand3. Step 3, PAREsnip Search the miRNA-mRNA pairs by PAREsnip m-way search tree. Step 4, hypothesis testing according  to PAREsnip Calculate p-value by PAREsnip. Step 5, for the site(p<0.05), plot t-plot figure according to the category of site using CleaveLand3 .
Genome_build: ASM41409v2
Supplementary_files_format_and_content: [density.txt] abundance measurements.  The first file contains degradome_density of the sample. 1. Each entry begins with the gene name in a FASTA-like header; 2. The next line in each entry begins with a comment symbol '#' and then gives the total size of the transcript (in nucleotides); 3. the remaining lines in each entry report the number of 5' ends at each occupied position, and these are tab-delimited lines; columns are: 1) nucleotide position within transcript (1-indexed), 2) total number of 5' ends at that position, 3) repeat-normalized number of 5'ends at that position, 4) total number of uniquely mapped 5' ends at that position, 5) The "category" of that position.
Supplementary_files_format_and_content: [miRNAs.txt] target genes.  The second file contains target genes of miRNAs of the sample. 1. The name of miRNA; 2.The name of target gene; 3.Cleavage site: Nucleotide number from 5’ end of cDNA; 4. Cleavage location; 5.Score: mapping score of mRNA-miRNAs pairwise; 6. Category: the "category" of this cleave site; 7. TP100M: Transcripts per 100 million; 8.P-value ≤0.05 using Cleaveland pipeline.
Supplementary_files_format_and_content: [cDNA.txt] degradome tags and quantity of degradome tags.  The third file shows tag sequences perfectly matching reference mRNA sequence of the sample. 1. Tag sequence Id; 2. cDNA ID or EST ID or mRNA ID; 3. Start position; 4. End position; 5. Strand; 6. Tag sequence; 7. Count of this tag sequence.

organism: Morus alba

source_name: drought-stressed mulberry

growth_protocol: The experimental mulberry species, ‘Yu711’ were grown under standard conditions (25°C day/20°C night, 14-h photoperiod) in the greenhouse at the National Mulberry Gene Bank in Zhenjiang, Jiangsu, China. The mulberry shoots were grafted to mulberry stock and were planted in 35 cm diameter pots containing loam soil, each pot containing one seedling.

extract_protocol:
Total RNA of each sample was extracted by Trizol (Life Technology) according to the manufacturers' instruction, and  for one group, the RNA from different time points under the same treatment were mixed together with equal amount.
In brief, poly (A) RNA was extracted from approximate 200 μg of total RNA using the Oligotex mRNA mini kit (Qiagen). A 5’-RNA adapter containing a Mme I recognition site in its 3’termini was ligated to the poly (A) RNA possessing a free 5’-monophosphate by T4 RNA ligase (Takara). The ligation products were purified using the Oligotex mRNA mini kit and reverse transcribed using Oligo (dT)18 primer via Superscript II reverse transcriptase (Invitrogen). The first-strand cDNA was amplified for 5 cycles using Ex Taq DNA Polymerase (Takara) and the PCR products were digested with Mme I. Next, the digested products were then ligated to a 3’-double-stranded DNA adaptor using T4 DNA ligase (Takara), gel purified and amplified by PCR for 20 cycles. The final PCR products were gel purified and subjected to SE51 sequencing by Illumina HiSeq2000

treatment_protocol: The grafted plants were randomly divided into two groups when the winter buds had grown to about 20 cm in length. One group was used as untreated control, and the other was treated with drought stress for 5 d, 10 d and 15 d. Each group contained three repeat individuals. For material harvest, young leaves were collected from the same position of different groups at different treated time points respectively and immediately frozen in liquid nitrogen and stored at -80℃ for RNA extraction.

tag: stress

value: drought

tag: tissue

value: young leaves

tag: growth condition

value: greenhouse


title: Morus alba L. control mulberry degradome

source_name: control mulberry

value: standard conditions


title: ESC_WT_2

value: wild type


title: ESC_KO_1

value: Phf8 KO


title: ESC_WT_1


title: KO_INP_2

data_processing:
Sequencing reads were mapped to the mouse genome (mm9 build) using the aligner bwa.
Input -normalized enrichment wig files were calculated using the R package SPP (Kharchenko, et al. 2008) as smoothed maximum likelihood log2 enrichments.
Supplementary_files_format_and_content: SPP-generated input-normalized enrichment wig files

source_name: Phf8 KO_cortical neuronal chromatin_None

extract_protocol:
Neuronal nuclei were FACS sorted from the neocorteces of WT and KO mice. Native chromatin was fragmented by MNase digest and ChIPed with the indicated antibody
Libraries were constructed with the Illumina TruSeq ChIP prep kit following the manufacturer's protocol

value: Neocortex

tag: chip antibody


title: WT_INP_2

source_name: wild type_cortical neuronal chromatin_None


title: WT_H3K4me1_2

source_name: wild type_cortical neuronal chromatin_H3K4me1

tag: chip antibody vendor

value: Abcam

tag: chip antibody cat. #

tag: chip antibody lot #


title: KO_H3K4me1_2

source_name: Phf8 KO_cortical neuronal chromatin_H3K4me1

tag: strain background


title: KO_H3K4me3_2

source_name: Phf8 KO_cortical neuronal chromatin_H3K4me3

tag: genotype/variation

value: Millipore


title: MAS-2 RNA-seq

city: Chengdu

first: Xinquan

last: Zhang

state: Sichuan

address: NO. 211 Huimin Road   Wenjiang District

department: Department of Grassland Science

institute: Sichuan Agricultural University

data_processing:
De novo assembly of RNA-seq was conducted by using Trinity (http://trinityrnaseq.sourceforge.net/).
The unigenes were generated by Trinity modules and the processes of sequence splicing and redundancy removing were employed.
Distinct clustering was performed to cluster the assembled unigenes transcript sequences to identify the same gene or homolog using a hierarchical clustering approach involving TGICL-CAP3 and CD-HIT20.
The alignment results were used to decide the sequence direction of the unigenes using TGICL 2.1 (http://sourceforge.net/projects/tgicl/files/tgicl%20v2.1/) and Phrap Release 23.0 (http://www.phrap.org/).
Genome_build: De novo assembly
Supplementary_files_format_and_content: FPKM values; Unigenes of each sample in fasta format

organism: Lolium multiflorum

source_name: susceptible  L. multiflorum  lines

extract_protocol:
Total RNA of each sample was extracted using the Trizol reagent (Agilent RNA 6000 nano Reagents) according to the manufacturer’s instructions
A total of 5 ug of total RNA per sample was used to construct the cDNA library using the NEB Next Ultra TM RNA Library Prep Kit for Illumina (New England Biol-abs, USA).Initially, the total RNA sample was digested using DnaseⅠ (NEB) and purified by oligo-dT breads (Dynabeads mRNA purification kit, Invitrogen). Following purification, the ploy (A)-containing mRNA was fragmented into 200 - 250 bp small pieces using fragment buffer (Ambion), and the first-strand cDNA was synthesis using the N6 primer, First Strand Master Mix and Super Script II reverse transcription (Invitrogen).

tag: phenotype

value: susceptible line

tag: sampling period

value: 20-day-old seedlings

value: whole plant

tag: treated with

value: drought for 2 hrs


title: Sox21a-/-_rep1

city: Beijing

first: huanwei

last: huang

address: 7 Science Park Rd., ZGC life science park

institute: NIBS

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to BDGP5 genome using TopHat v2.0.10 with default parameters
Cufflinks v2.2.1 was run for relative quantitation of genes expression and differential expression analysis.
Genome_build: Ensemble Drosophila melanogaster BDGP5
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.

organism: Drosophila melanogaster

source_name: intestinal progenitor cells_Sox21a-/-

extract_protocol:
Drosophila midgut were removed and digested using Elastase (Sigma, cat. no. E0258). The progenitor cells labeled with green fluorescent protein (GFP) were sorted using FACSAria II cell sorter (Becton Dickinson) and RNA was harvested using the Direct-zol™ RNA MiniPrep(Zymo research , cat. no. E0258). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 50 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: midgut

tag: cell type

value: intestinal progenitor cells

tag: age

value: adult stage day 10

tag: genotype


title: Esg_rep1

source_name: intestinal progenitor cells_wildtype

value: Wild type


title: Esg_rep3


title: KO_H3K9me2_2

source_name: Phf8 KO_cortical neuronal chromatin_H3K9me2


title: Sox21a-/-_rep2


title: Esg_rep2


title: Esg_rep4


title: Sox21a-/-_rep3


title: KO_H3K4me3_1


title: WT H3K9me2_2

source_name: wild type_cortical neuronal chromatin_H3K9me2


title: WT H3K27me2_2

source_name: wild type_cortical neuronal chromatin_H3K27me2


title: WT H3K27me2_1


title: MAD-1 RNA-seq

source_name: tolerant L. multiflorum lines

value: resistant line

value: drought for 1 hr


title: WT_H3K4me3_2

source_name: wild type_cortical neuronal chromatin_H3K4me3


title: KO_H3K27me2_2

source_name: Phf8 KO_cortical neuronal chromatin_H3K27me2


title: KO_H3K27me2_1


title: KO_H3K9me2_1


title: KO_INP_1


title: MAS-0 RNA-seq

value: none (control)


title: MAS-1 RNA-seq


title: KO_H3K4me1_1


title: WT_H3K4me3_1


title: MAD-2 RNA-seq


title: WT-2

city: Shanghai

first: Xianghua

last: Yi

address: NO.389, Xincun Rd, Putuo distrct

department: pathology

institute: Tongji hospital

data_processing:
Illumina Casava 1.8.2 software used for basecalling
Sequenced reads were trinned for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to the mouse reference genome (GRCm38, Ensembl build) using Tophat version 2.0.12, yielding an average mapping rate of 90.3 ± 2.3%.
Gene expression levels, which represented as fragments per kilo-base per million mapped reads (FPKM), were obtained for 27180 genes/transcripts annotated using Ensemble release 67.
Genome_build: GRCm38
Supplementary_files_format_and_content: csv file with FPKM values.

source_name: mice bone marrow cells

extract_protocol:
LSCs of AML murine model were sorted by FACS, and RNA was harvested using Trizol reagent.  library construction following standard TruSeq RNA sample preparation v2 protocol (Illumina).
library construction following standard TruSeq RNA sample preparation v2 protocol (Illumina).

tag: strain

value: bone marrow

value: LSC

value: 6 weeks


title: KO-1


title: ovaries

first: Wei

last: Peng

address: Shizishan Street No. 1, Hongshan District, Wuhan,Hubei  430070, P.R. China

data_processing:
The raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats.
Unique sequences with length in 18~25 nucleotide were mapped to specific species precursors in miRBase 20.0 by BLAST search to identify known miRNAs and novel 3p- and 5p- derived miRNAs. Length variation at both 3’ and 5’ ends and one mismatch inside of the sequence were allowed in the alignment. The unique sequences mapping to specific species mature miRNAs in hairpin arms were identied as known miRNAs. The unique sequences mapping to the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3p derived miRNA candidates. The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 20.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations. The above two we defined as known miRNAs. The unmapped sequences were BLASTed against the specific genomes, and the hairpin RNA structures containing sequences were predicated from the flank 120nt sequences using RNAfold software (http://rna.tbi.univie.ac. at/cgi-bin/RNAfold.cgi).
The criteria for secondary structure prediction were: (1) number of nucleotides in one bulge in stem (<=12) (2) number of base pairs in the stem region of the predicted hairpin (>=16) (3) cutoff of free energy (kCal/mol <=15) (4) length of hairpin (up and down stems + terminal loop >=50) (5) length of hairpin loop (<=200). (6) number of nucleotides in one bulge in mature region (<=4) (7) number of biased errors in one bulge in mature region(<=2) (8) number of biased bulges in mature region (<=2) (9) number of errors in mature region (<=4) (10) number of base pairs in the mature region of the predicted hairpin (>=12) (11) percent of mature in stem (>=80).
Genome_build: ASM78921v1

organism: Bactrocera dorsalis

source_name: tissues

extract_protocol:
12-day old adult mature female and male adults were dissected. The gonads and the rest adult samples were immediately stored in RNAlater® Solution (Ambion). Total RNAs were extracted using Trizol reagent (Invitrogen, CA, USA). Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with total RNA for the construction of sequencing libraries.
Small RNA libraries were generated from the two samples using the Illumina Truseq Small RNA Preparation kit according to Illumina’s TruSeq Small RNA Sample Preparation Guide. The purified cDNA library was used for cluster generation on Illumina’s Cluster Station and then sequenced on Illumina GAIIx following vendor’s instruction for running the instrument. Raw sequencing reads were obtained using Illumina’s Sequencing Control Studio software version 2.8 (SCS v2.8) following real-time sequencing image analysis and base-calling by Illumina's Real-Time Analysis version 1.8.70 (RTA v1.8.70). A proprietary pipeline script, ACGT101-miR v4.2 (LC Sciences), was used for sequencing data analysis. Degradome library was constructed following with methods previously described by German et al.(2008).CleaveLand3.0 and LC Science’s ACGT301-DGEv1.0 program were used to detect potentially sliced targets of known and novel miRNA.

value: 12-day old adult


title: water deficit stress for 1 d

city: Pyeongchang

first: Pil Joong

last: Chung

state: Gangwondo

address: Pyeongchang daero 1447

department: Crop Biotechnology Institute

country: South Korea

institute: Seoul National University

description: water deficit stress for 1 d

data_processing:
Single-end seq were generated and raw seq reads were trimmed to removed adaptor seq, and those with a quality lower than Q20 were removed using the clc quality trim software (CLCBIO).
Duplicate paired short reads were removed using FastUniq.
All reads were assembled with the clc_ref_assmble 6 (version 4.06) program, using annotated gene and noncoding RNA seq from the rapdb (http://rapdb.dna.affrc.go.jp) and ncRNA (http://www.ncrna.org) databases.
genome_build: IRGSP-1.0

organism: Oryza sativa

source_name: 5-week old wild type rice

growth_protocol: Rice was germinated on MS media at 28C for 4 days, and transplanted into soil pots and grown in a greenhouse.

extract_protocol:
Total RNA was extracted from rice leaves using Trizol reagent (Invtrogen) and purified an RNeasy Mini Kit (Qiagen).
A modified TruSeq method was used to construct strand-specific RNA-seq libraries with differnet ibdex primers.
barcode: CGATGT

treatment_protocol: Multiple pots of each rice were divided into 2 sets, one for drought and one for well-watered treatment conditions. Five weeks after transplanting to soil, water was withheld, and the soil water content in each pot was adjusted to approximately 75%. Soil moisture was monitored during the drought treatment using a Soil Moisture Sensor SM150 (Delta-T Devices, UK)

tag: ecotype

value: Illmi

tag: tissue types

value: leaves

tag: tissue age

value: 5 weeks


title: WT H3K9me2_1

value: wild type

tag: tissue

value: Neocortex

value: Abcam


title: water deficit stress for 2 d

description: water deficit stress for 2 d

extract_protocol:
Total RNA was extracted from rice leaves using Trizol reagent (Invtrogen) and purified an RNeasy Mini Kit (Qiagen).
A modified TruSeq method was used to construct strand-specific RNA-seq libraries with differnet ibdex primers.
barcode: TTAGGC


title: testes


title: anti-CD40L Spleen IgM 2

city: Atlanta

first: Caitlin

middle: Darcy

last: Bohannon

state: GA

address: 210 Dowman Drive

department: Immunology

institute: Emory University

description: Ig heavy chain

data_processing:
Merge fasta and qual files
Split libraries
Analyzed with IGMT High V-Quest
THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM®
Genome_build: N/A
Supplementary_files_format_and_content: see README.txt

source_name: anti-CD40L Spleen IgM 2

extract_protocol:
RNA from these cells was isolated with the RNeasy kit (Qiagen)
Library transcribed into cDNA with ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis (Invitrogen)

treatment_protocol: Total plasma cells (CD138+B220-) were isolated via cell sorting from the marrow and spleens of C57/BL/6 mice 1 month post-immunization with NP22CGG

tag: barcode

value: GGCATATTAG

value: CD138+B220- Plasma cells

tag: time point

value: 1 month post NPCG immunization

tag: treatment

value: Treated with anti-CD40L


title: males


title: anti-CD40L Marrow IgM 1

description: Ig heavy chain

source_name: anti-CD40L Marrow IgM 1

value: GCCAACATTA


title: well-watered control

description: control leaves

extract_protocol:
Total RNA was extracted from rice leaves using Trizol reagent (Invtrogen) and purified an RNeasy Mini Kit (Qiagen).
A modified TruSeq method was used to construct strand-specific RNA-seq libraries with differnet ibdex primers.
barcode: ATCACG


title: WT-1


title: anti-CD40L Marrow IgM 2

description: Ig heavy chain

source_name: anti-CD40L Marrow IgM 2

value: GGTTCATTAG


title: KO-2


title: MAD-0 RNA-seq

value: resistant line

value: 20-day-old seedlings

value: whole plant

value: none (control)


title: WT_H3K4me1_1

source_name: wild type_cortical neuronal chromatin_H3K4me1

tag: chip antibody

tag: chip antibody vendor

tag: chip antibody cat. #

tag: chip antibody lot #


title: females

value: 12-day old adult


title: Seb1_Input_2_ChIP-Seq

city: Oxford

first: Beth

last: Watts

address: South Parks Road

country: United Kingdom

institute: University of Oxford

description:
h+, ura4-Δ18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+, seb1-3HA-TAP::kan MX
genomic DNA

data_processing:
ChIP-seq: Single end reads with a Phred score of less than 20 were removed using the the program Trimmomatic (version 0.36) and the remaining reads were aligned to the S. pombe genome (ASM294v2.25 from Pombase.org) using the short read aligner Bowtie (version 2.2.6). The BEDTools toolset was used to obtain coverage tracks.
genome build: S. pombe genome version ASM294v2.28
processed data files format and content: ChIP-Seq: the data is provided as bigwig  files

organism: Schizosaccharomyces pombe

source_name: Yeast cell extract

growth_protocol: S. pombe were grown in YES medium to exponential phase at 30°C

extract_protocol:
Cells were grown to mid-log phase and crosslinked using 11% formaldehyde. Cells were lysed by vortexing with glass beads and chromatin was extracted by two consecutive ultra-centrifugation steps. The DNA was sheared by sonication and DNA was extracted by phenol:chloroform.
Libraries were generated using the NEBNext fast DNA library Prep set for Ion Torrent (E6270).

value: h+, ura4-delta18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+, seb1-3HA-TAP::kan MX

tag: method

value: ChIP-Seq


title: Seb1_IP_2_ChIP-Seq

description:
h+, ura4-Δ18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+, seb1-3HA-TAP::kan MX
crosslinked DNA

extract_protocol:
Cells were grown to mid-log phase and crosslinked using 11% formaldehyde. Cells were lysed by vortexing with glass beads and chromatin was extracted by two consecutive ultra-centrifugation steps. The DNA was sheared by sonication, Pcf11-TAP was IP-ed using IgG agarose and DNA was extracted by phenol:chloroform.
Libraries were generated using the NEBNext fast DNA library Prep set for Ion Torrent (E6270).


title: WT_INP_1

source_name: wild type_cortical neuronal chromatin_None

tag: strain background

tag: genotype/variation


title: anti-CD40L Spleen IgM 1

description: Ig heavy chain

source_name: anti-CD40L Spleen IgM 1

value: GAATTCATAT

tag: cell type


title: NPCG Marrow IgM 1

description: Ig heavy chain

source_name: NPCG Marrow IgM 1

value: AGCACTGTAG


title: Seb1_IP_1_ChIP-Seq

tag: genotype


title: N-minus 48h rep2

city: Seoul

first: Han

middle: Min

last: Woo

address: Hwarangno 14-gil 5, Seongbuk-gu

department: Clean Energy Research Center

institute: Korea Institute of Science and Technology

data_processing:
base-calling - Illumina MiSeq
alignment - Bowtie 2.0
generation of normalized abundance measurements  - EdgeR
Genome build: ASM1252v1
Supplementary_files_format_and_content: Se7942-N-starv2016.txt reports normalized RPKMs

organism: Synechococcus elongatus PCC 7942 = FACHB-805

source_name: N-minus 48h

growth_protocol: Synechococcuse longatuse PCC 7942 strains were grown at 30 °C temperature while shaking under constant illumination light (100μE m-2 s-1) provided in BG-11 medium.

extract_protocol:
5 mL of Trizol reagent (California, Carlsbad, USA) was added to the cyanobacterial cell pellet under liquid nitogen. To break the cell wall, the quick frozen cell was grinded using a mortar. After addition 200 ul of bromo-chloro-propane and incubation at room temperature, the extraction mixture was centrifuged to separate the phase. Retrieved aqueous phase was   mixed with the equal volume of isopropanol, incubated 5min at room temperature and centrifuged to concentrate the precipitated RNA. The RNA pellet was washed using 75% ethanol, air dried and finally dissolved in RNA storage solution
Total RNA (10 µg) from each sample was used as a starting material to prepare sequencing libraries. The mRNA was purified with the Dynabeads® mRNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). The resulting mRNA samples were processed for the sequencing libraries using the Illumina mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA) by the manufacturer’s protocols.

treatment_protocol: For nitrogen starvation experiments, we used up change a medium from BG-11 medium to lacking combined nitrogen (BG-11 N-: no added NaNO3; this medium corresponded to N-). Briefly, cells were grown in 6 ml BG-11(N+) in for 24hours

tag: strain

value: N-minus

tag: time


title: NPCG Spleen IgM 1

description: Ig heavy chain

source_name: NPCG Spleen IgM 1

value: AGACGCACTC


title: Control 24h rep2

source_name: Control 24h

value: Control


title: WT_IP_2_ChIP-Seq

description:
h+, ura4-Δ18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+
crosslinked DNA

value: h+, ura4-delta18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+


title: N-minus 48h rep1


title: NPCG Marrow IgG 2

description: Ig heavy chain

source_name: NPCG Marrow IgG 2

value: ACGCTCGACA

value: CD138+B220- Plasma cells

value: 1 month post NPCG immunization


title: WT_Input_1_ChIP-Seq

description:
h+, ura4-Δ18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+
genomic DNA


title: NPCG Spleen IgM 2

description: Ig heavy chain

source_name: NPCG Spleen IgM 2

value: ATCACTGACA


title: small RNA-seq_ade6-TNI wt

status: Public on Feb 13 2017

city: Basel

first: Fabio

last: Mohn

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

description: ade6-TNI wt

data_processing:
Samples were pooled and sequenced on an Illumina HiSeq 2500 instrument.
Demultiplexing and fastq conversion were performed with bcl2fastq2 v1.17.
Illumina 3' adaptors were trimmed using cutadapt with parameters -a TGGAATTCTCGGGTGCCAAGG --discard-untrimmed -m 18.
Fastq files were aligned to the S. pombe genome using Bowtie 1.0.0 with parameters -M 1 -v 0  --best --strata.
Bowtie sam out put was converted to bam using samtools (v1.3.1). Bam files were converted to bed using bedtools bamtobed (v2.25.0), Bed files were used to calculate strand-specific coverage using bedtools genomecov (v2.25.0) and resulting bedgraph files were converted to bigWig format using bedGraphToBigWig (v4).
Genome_build: S. pombe ASM294v2.24
Supplementary_files_format_and_content: bigWig files containing normalized (to 1 mio genome mapping reads per library) genome mapping read counts for visualization in UCSC or IGV.

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted non-splicable antisense cox4 intron in the ade6 gene in wt background.

extract_protocol:
Total RNA was isolated from exponentially growing cells using hot phenol. The RNA was fractionated using RNeasy Midi columns (Qiagen). The flow-through fraction was precipitated and aliquots of 25 ug were separated by 17.5% PAGE to isolate the 18–28-nucleotide fraction.
Libraries were prepared using the Illumina TruSeq small RNA kit according to manufacturers instructions. For barcodes, see sample characteristics.

value: h+ leu1-32 ura4-D18 ade6-target2 nmt1+::ura4-hp+-nat

tag: molecule subtype

value: size selected small RNA from whole cell lysate

tag: illumina barcode

value: TGACCA


title: small RNA-seq_ade6-5'INT wt

description: ade6-5'INT wt

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted cox4 intron in the ade6 5'UTR in wt background.

value: h- leu1-32 ura4-D18 ade6-target4 nmt1+::ura4-hp+-nat

value: GCCAAT


title: small RNA-seq_ade6-5'INT leo1D

description: ade6-5'INT leo1D

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted cox4 intron in the ade6 5'UTR in leo1 mutant background.

value: h- leu1-32 ura4-D18 ade6-target4 nmt1+::ura4-hp+-nat leo1D::kan

value: ACAGTG


title: small RNA-seq_ade6-5'TNI leo1D

description: ade6-5'TNI leo1D

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted non-splicable antisense cox4 intron in the ade6 5'UTR in leo1 mutant background.

value: h- leu1-32 ura4-D18 ade6-target5 nmt1+::ura4-hp+-nat leo1D::kan

value: CAGATC


title: NPCG Marrow IgM 2

description: Ig heavy chain

source_name: NPCG Marrow IgM 2

value: GTTAACACAT


title: N-plus 48h rep1

source_name: N-plus 48h

value: N-plus


title: small RNA-seq_ade6-5'TNI wt

description: ade6-5'TNI wt

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted non-splicable antisense cox4 intron in the ade6 5'UTR in wt background.

value: h- leu1-32 ura4-D18 ade6-target5 nmt1+::ura4-hp+-nat

value: ACTTGA


title: N-plus 48h rep2


title: Seb1_Input_1_ChIP-Seq

value: h+, ura4-delta18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+, seb1-3HA-TAP::kan MX

value: ChIP-Seq


title: water deficit stress for 3 d

description: water deficit stress for 3 d

source_name: 5-week old wild type rice

extract_protocol:
Total RNA was extracted from rice leaves using Trizol reagent (Invtrogen) and purified an RNeasy Mini Kit (Qiagen).
A modified TruSeq method was used to construct strand-specific RNA-seq libraries with differnet ibdex primers.
barcode: TGACCA

tag: ecotype

value: Illmi

tag: tissue types

value: leaves

tag: tissue age

value: 5 weeks


title: small RNA-seq_ade6-TNI leo1D

description: ade6-TNI leo1D

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted non-splicable antisense cox4 intron in the ade6 gene in leo1 mutant background.

value: h- leu1-32 ura4-D18 ade6-target2 nmt1+::ura4-hp+-nat leo1D::kan

value: size selected small RNA from whole cell lysate

value: TTAGGC


title: small RNA-seq_ade6-INT wt

description: ade6-INT wt

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted cox4 intron in the ade6 gene in wt background.

value: h- leu1-32 ura4-D18 ade6-target1 nmt1+::ura4-hp+-nat

value: CGATGT


title: WT_Input_2_ChIP-Seq

source_name: Yeast cell extract

value: h+, ura4-delta18, leu1-32, ade6-M216, imr1R(NcoI)::ura4+


title: WT_IP_1_ChIP-Seq


title: small RNA-seq_ade6-INT leo1D

description: ade6-INT leo1D

source_name: small RNA sequencing from strain expressing siRNAs targeting an inserted cox4 intron in the ade6 gene in leo1 mutant background.

value: h- leu1-32 ura4-D18 ade6-target1 nmt1+::ura4-hp+-nat leo1D::kan

value: ATCACG


title: NM-K rep3

city: Madison

first: Jean-Michel

last: Ané

state: WI

address: 1550 Linden Drive

department: Bacteriology

institute: University of Wisconsin-Madison

data_processing:
RNA-sequencing (RNA-Seq) data were processed to obtain differentially expressed genes (DEGs) using TopHat2-SE, Cufflinks2, Cuffmerge2 and Cuffdiff2 applications from the iPlant Collaborative platform (http://www.iplantcollaborative.org).
The DESeq Bioconductor package version 3.3 for R (Anders and Huber, 2010) was used for the analysis of DEGs. Cuffdiff2 sequencing data were analyzed using the cummeRbund package to generate principal component and multidimensional scaling analyses on biological and technical replicates.
Genome_build: Mt4.0v1
Supplementary_files_format_and_content: tab-delimited text file displaying FPKM values for each condition

organism: Medicago truncatula

source_name: NM-K

extract_protocol:
Frozen root tissues were ground in liquid nitrogen, and total RNA was extracted from the resulting powder using PureLink® RNA Mini Kit (Thermofisher), and treated with TURBOTM DNase (Thermofisher). RNA quality was assessed using the Agilent 2100 BioAnalyzer technology (Agilent Technologies, Santa Clara, CA, USA).
Six micrograms of total RNA were used to construct polyA selection libraries using the Illumina TrueSeq RNA Sample Preparation kit; 100-nt single-end reads were obtained on an Illumina Hiseq2000 platform using TruSeq v3 (University of Wisconsin Biotechnology Center DNA Sequencing Facility, USA).

tag: host genotype

value: Jemalong A17

tag: inoculation

value: control

tag: growth protocol

value: -K

tag: tissue

value: roots


title: NM-K rep4


title: Control 24h rep1

tag: treatment

value: Control


title: NM-K rep2


title: NPCG Marrow IgG 1

description: Ig heavy chain

source_name: NPCG Marrow IgG 1

tag: barcode

value: ACGAGTGCGT

value: CD138+B220- Plasma cells

tag: time point

value: 1 month post NPCG immunization


title: AM-K rep3

source_name: AM-K

value: Rhizophagus irregularis


title: NM+K rep1

source_name: NM+K

value: +K


title: NM+K rep3


title: NM+K rep4


title: AM-K rep1


title: AM+K rep1

source_name: AM+K


title: AM-K rep2


title: AM+K rep3


title: parp1parp2-flg22

city: Riverside

first: Brandon

last: Le

address: 3401 Watkins Drive

department: Institute for Integrative Genome Biology

institute: University of California, Riverside

data_processing:
Raw reads were processed by first trimming 3’ adapter sequence using custom Perl script. Reads without 3’ adapter sequence or less than 18 nucleotides after trimming were discarded from further analysis.
Trimmed reads were aligned against a custom database containing rRNA, tRNA, snoRNA, and snRNAs using Bowtie 0.12.8 to filter out rRNA reads.
Non-aligned non-rRNA reads were subsequently aligned to the Arabidopsis genome (TAIR10) using Bowtie 0.12.8 (-v 0 -a -m 1000). Genome-aligned reads were used for all downstream analysis.
Genome_build: TAIR10
Supplementary_files_format_and_content: miRNA counts for each sample were generated using Bedtools and annotations from miRBase v21.

organism: Arabidopsis thaliana

source_name: 10 day old seedlings

growth_protocol: Ten-day-old seedlings grown on ½ Murashige and Skoog (MS) plates with 0.8% agar

extract_protocol:
RNA was extracted with TRIzol reagent (Life Technologies, USA, 15596018)
Small RNA fragments (~15–30 nucleotides) were selected and recovered from 10 µg of total RNAs by 15% urea-polyacrylamide gel electrophoresis. Libraries were made using the NEBNext Small RNA Library Prep kit (NEB, USA) according to manufacturer’s instructions. Briefly, 3' and 5' adapters were ligated sequentially to the small RNAs. Ligated small RNAs were converted to cDNA by reverse transcription followed by PCR amplification for 15 cycles. The barcoded libraries were sequenced in one lane on the Illumina Hiseq2000.

treatment_protocol: Ten-day-old seedlings grown on ½ MS plates were transferred to 2 ml of H2O in a 6-well plate one day before 100 nM flg22 treatment.

value: 100 nM flg22


title: WT-flg22

value: WT


title: NM-K rep1


title: parg1-flg22


title: DCL1-9

first: Zhenfei

last: Sun

address: Dongchuan Road 500

institute: Shanghai Jiaotong University

data_processing:
base-calling performed using HCS,RTA and OLB 1.9.4
Data were filtered by the following steps:  1) Getting rid of low quality reads (The criteria for this is listed in the explanation of meaning of each row in the result table)  2) Getting rid of reads with 5' primer contaminants  3) Getting rid of reads without 3' primer  4) Getting rid of reads without the insert tag  5) Getting rid of reads with poly A  6) Getting rid of reads shorter than 18nt
Map the small RNA tags to genome by SOAP2.20 to analyze their expression and distribution on the genome.  Program and Parameters:  soap -v 0 -r 2 -M 0 -a clean.fa -D ref_genome.fa.index -o match_genome.soap
Alignments to mirbase, Genbank, Rfam performed by blastn: blastall -p blastn -F F -e 0.01
The small RNA reads were trimmed for adaptor sequence using Perl scripts and mapped to miRNA hairpin sequences downloaded from mirRBase version 15.0 using the Bowtie program
genome build: TAIR10
Supplementary_files_format_and_content: Differential expression data of miRNAs between cdf2 mutant and WT in WT-CDF2 miR_count.sig, or differential expression data of miRNAs between dcl1-9 mutant and WT in WT-DCL1-9-miR_count.sig files."expressed" means TPM (Transcripts per million)

source_name: seedling

growth_protocol: plants were grown in soil at 16 hr light/8 hr dark photoperiod

extract_protocol:
Small RNAs were isolated using mirVana™miRNA Isolation Kit (Ambion, AM1561)
Small RNA libraries were prepared and sequenced by Illumina high-throughput sequencing

treatment_protocol: no treatment

tag: age

value: Col

tag: genotype/variation

value: DCL1 mutnat


title: WT

data_processing:
base-calling performed using HCS,RTA and OLB 1.9.4
Data were filtered by the following steps:  1) Getting rid of low quality reads (The criteria for this is listed in the explanation of meaning of each row in the result table)  2) Getting rid of reads with 5' primer contaminants  3) Getting rid of reads without 3' primer  4) Getting rid of reads without the insert tag  5) Getting rid of reads with poly A  6) Getting rid of reads shorter than 18nt
Map the small RNA tags to genome by SOAP2.20 to analyze their expression and distribution on the genome.  Program and Parameters:  soap -v 0 -r 2 -M 0 -a clean.fa -D ref_genome.fa.index -o match_genome.soap
Alignments to mirbase, Genbank, Rfam performed by blastn: blastall -p blastn -F F -e 0.01
The small RNA reads were trimmed for adaptor sequence using Perl scripts and mapped to miRNA hairpin sequences downloaded from mirRBase version 15.0 using the Bowtie program
genome build: TAIR10
Supplementary_files_format_and_content: Differential expression data of miRNAs between cdf2 mutant and WT in WT-CDF2 miR_count.sig, or differential expression data of miRNAs between dcl1-9 mutant and WT in WT-DCL1-9-miR_count.sig files. "expressed" means TPM (Transcripts per million)


title: parg1

value: no treatment

tag: genotype


title: CDF2

data_processing:
base-calling performed using HCS,RTA and OLB 1.9.4
Data were filtered by the following steps:  1) Getting rid of low quality reads (The criteria for this is listed in the explanation of meaning of each row in the result table)  2) Getting rid of reads with 5' primer contaminants  3) Getting rid of reads without 3' primer  4) Getting rid of reads without the insert tag  5) Getting rid of reads with poly A  6) Getting rid of reads shorter than 18nt
Map the small RNA tags to genome by SOAP2.20 to analyze their expression and distribution on the genome.  Program and Parameters:  soap -v 0 -r 2 -M 0 -a clean.fa -D ref_genome.fa.index -o match_genome.soap
Alignments to mirbase, Genbank, Rfam performed by blastn: blastall -p blastn -F F -e 0.01
The small RNA reads were trimmed for adaptor sequence using Perl scripts and mapped to miRNA hairpin sequences downloaded from mirRBase version 15.0 using the Bowtie program
genome build: TAIR10
Supplementary_files_format_and_content: Differential expression data of miRNAs between cdf2 mutant and WT in WT-CDF2 miR_count.sig, or differential expression data of miRNAs between dcl1-9 mutant and WT in WT-DCL1-9-miR_count.sig files.

value: CDF2 mutant


title: Beaf32_29C_3

city: Berkeley

first: Shaked

last: Afik

address: Stanley Hall

department: Computational Biology Graduate Group

institute: UC Berkeley

description:
To down regulate beaf-32, we generated flies expressing a UAS-RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. RNA was collected at 29C
beaf.32.counts.txt

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to dm3 whole genome and transcriptome using STAR (for 3’-sequencing using 100bp) or tophat2 (all other samples)
Beaf32 experiment: Read count was calculated using ESAT. Full length RNA-seq and 3’-seq using 100bp were only used for validation of Exo-seq and RNAseH-seq and thus were not processed further.
Genome_build: dm3
Supplementary_files_format_and_content: Expression matrix of the read counts for each genes in wild type and beaf32 knockdown flies

molecule: polyA RNA

source_name: 3' RNA-Seq (without RNase H treatment)

growth_protocol: BEAF32 experiment: To down regulate beaf-32, we generated flies expressing a UAS-RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. Flies were reared at 25°C on a standard diet (yeast: 38gr/L, Yellow corn mill: 91gr/L, agar: 10gr/L, molasses: 8.7% v/v, propanoic acid (BioLab): 0.9% v/v, Tegasept solution (Sigma-Aldrich; 300g/L in EtOH (BioLab)): 0.8% v/v)). After eclosion flies were kept in 12-h light:12-h dark cycles at 25°C. Newborn flies were entrained for three days in 12:12 light: dark conditions at different temperatures (18°C, 29°C). Full length RNA-seq and 3’-seq using 100bp: CS fly heads (1 ml) were collected at ZT3 and homogenized in 3 mL of NEB buffer (10 mM HEPES, pH 8.0, 10 mM NaCl, 0.1 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 0.5% Tergitol NP-10, 0.5 mM spermidine, 0.15 mM spermine plus protease inhibitor tablets [Roche])

extract_protocol:
BEAF32 experiment: RNA was extracted from their heads using TRI Reagent, Sigma Aldrich. Full length RNA-seq and 3’-seq using 100bp: RNA was extracted using Trizol (Invitrogen).
BEAF32 experiment: (Dnase treated) RNA was fragmented in the presence of 10XFastAP buffer (ThermoFisher Scientific), at 94°C for 3 minutes, and placed on ice. RNA was then dephosphorylated with FastAP, cleaned (2X SPRI) and ligated to linker1. Library preparation was continued as described for Exo-seq, starting from linker1 3’ ligation. Full length RNA-seq: Full-length RNA libraries were done similarly to the described 'RNAseH- samples', with a difference in the order of the different steps; First, RNA was PolyA+ selected, then fragmented (using Mg-based fragmentation), cleaned (2.5X SPRI cleanup), FastAP treated, cleaned (Silane cleanup) and continued in library preparation (starting at first ligation of 3’ adapter). 3’-seq using 100bp: Library preparation was done as described for RNaseH- samples.

tag: strain

value: YW

value: head

value: beaf32 knockdown

tag: temperature tested


title: Beaf32_29C_2


title: Beaf32_29C_1


title: NM+K rep2

value: Jemalong A17

value: control


title: Beaf32_18C_2

description:
To down regulate beaf-32, we generated flies expressing a UAS-RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. RNA was collected at 18C
beaf.32.counts.txt


title: Beaf32_18C_1


title: YW_29C_3

value: wild type


title: Beaf32_18C_3


title: YW_29C_2


title: YW_18C_2


title: parp1parp2


title: YW_18C_1


title: WT


title: 3end_100bp_1

description: 3' end RNA without RNase H treatment sequenced with 100bp. First read starts with 8 bases of the barcode

source_name: 3' RNA-Seq (without RNase H treatment) using 100bp

value: Canton S


title: Full_length_25C_zt15

description: full length RNA-seq of fly heads at 25C, zt15

source_name: full length RNA-seq (without RNase H treatment)


title: Full_length_25C_zt3

description: full length RNA-seq of fly heads at 25C, zt3


title: CS1_H3K4me3 [ChIP-seq]

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were aligned to the genome using bowtie2
Peak calling was done using MACS
Genome_build: dm3
Supplementary_files_format_and_content: bed files of the peaks called by MACS from the ChIP-seq data

growth_protocol: CS fly heads (1 ml) were collected at ZT3 and homogenized in 3 mL of NEB buffer (10 mM HEPES, pH 8.0, 10 mM NaCl, 0.1 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 0.5% Tergitol NP-10, 0.5 mM spermidine, 0.15 mM spermine plus protease inhibitor tablets [Roche])

extract_protocol:
Homogenateswere placed in Bio-Spin columns (Bio-Rad) and centrifuged at 1000g for 4 min. Filtered homogenates were then centrifuged at 6000g for 10 min. The nuclei-containing pellets were resuspended in 1 mL of NEB and centrifuged at 20,000g for 20 min on sucrose gradients (0.65 mL of 1.6 M sucrose in NEB, 0.35 mL of 0.8 M sucrose in NEB). The pellet was resuspended in 1 mL of NEB and 11% formaldehyde (diluted in Schneider's media; Sigma) was added to a final concentration of 1%. Nuclei were cross-linked for 10 min at room temperature and quenched by adding 1/10 volume of 1.375 M glycine. The nuclei were collected by centrifugation at 6000g for 5 min. Nuclei were washed twice in 1 mL of NEB and resuspended in 450 μL of Sonication buffer (10 mM HEPES, pH 7.5, 2 mM EDTA, 1% SDS, 0.2% Triton X-100, 0.5 mM spermidine, 0.15 mM spermine) and sheared by sonication three times for 7 min using a Bioruptorsonicator (Diagenode) on ice. Sonicated samples were centrifuged at 15,000g for 10 min and frozen at −80°C in 150-μL aliquots. Of the sonicated chromatin, 25 µL was used as input control. The remaining 125 μL were diluted in 1.1 mL of IP buffer (50 mM HEPES/KOH, pH 7.6, 2 mM EDTA, 1% Triton, 0.1% deoxycholate). Antibody was added: 4 µg of anti-H3K4met3 antibody (abcam8580), and samples were rotated at 4 °C overnight Protein A/G-plus beads (Santa Cruz biotechnology) were blocked overnight in 0.1 mg/mL yeast tRNA and 1 mg/mL BSA in IP buffer. After overnight incubation, the beads were washed once in IP buffer, added to the chromatin/antibody mixture, and then incubated for an additional 2 h at 4°C.Beads were centrifuged at 1,000 rpm for 20s and were washed once in 1.5 mL of ChIP Wash buffer (50 mM HEPES-KOH, pH 7.6, 1 mM EDTA, 1% Triton, 0.1% deoxycholate, 0.1% Sarkosyl, 0.1% BSA, 0.5 M KCl). Beads were resuspended in 1 mL of ChIP Wash buffer and rotated for 30 min at 4°C. Beads were then washed once in Li Wash Buffer (10 mM Tris, pH 8.0, 0.25 M LiCl, 0.5% NP40, 0.5% deooxychoalte, 1 mM EDTA) and once in cold TE (pH 8.0) before being eluted with 150 μL of ChIP Elution buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM DTT, 0.1 mg/mL proteinase K). ChIP Elution buffer (150 μL) was also added to the input sample. Both IP and input samples were incubated for 2h at 37°C. The sepharose beads were removed from the IP samples, and cross-links were reversed by addition of 20µL 5M NaCl and incubation overnight at 65°C. DNA was isolated from the samples using phenol-chloroform (Sigma) extraction.
DNA library preparation was performed as described by Blecher-Gonen et al. : "High-throughput chromatin immunoprecipitation for genome-wide mapping of in vivo protein-DNA interactions and epigenomic states".

tag: growth temperature

tag: chip antibody

tag: chip antibody vendor

value: abcam

tag: chip antibody catalog #

tag: chip antibody lot #


title: RNAseH_25C_zt3

description: processed data file: RNsseH.merged.wig

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to dm3 whole genome and transcriptome using tophat2
peaks were called using our a stand alone java program build to analyze our novel sequencing method
Genome_build: dm3
Supplementary_files_format_and_content: All 3’ without RNaseH treatment data was divided to true and false based on whether or not the 3 last bases of the first read were adenines or not (in order to separate internal poly(A) tracts and poly(A) tails). TDF files were created by IGVtools with the parameters igvtools -count -w 5. Wig files were created using a customized python scripts, where for each genomic position we counted the number of read that started on this position (i.e. the first base of the read was aligned to this position). For every sequencing method (ExoCAGE, 3’-sequencing and RNaseH sequencing) the data from different temperatures and time points were merged together to create the processesed files.

source_name: 3’ RNase H-sequencing

growth_protocol: Drosophila Canton S (CS) wild type flies were reared at 25 °C on a standard diet (yeast: 38gr/L, Yellow corn mill: 91gr/L, agar: 10gr/L, molassas: 8.7% v/v, propanoic acid (BioLab): 0.9% v/v, Tegasept solution (Sigma-Aldrich; 300g/L in EtOH (BioLab)): 0.8% v/v)). Flies were kept in 12-h light:12-h dark cycles at 25 °C.

extract_protocol:
For RNA extraction, new-born flies were entrained for three days in 12:12 light:dark conditions at one of the different temperatures tested (18C, 25C or 29C) and collected at one of the time points (ZT3,ZT7,ZT9,ZT11,ZT15,ZT19,ZT21 or ZT23). RNA was extracted using Trizol (Invitrogen).
ExoCAGE: 50ng-1ug of total RNA was polyA selected using Oligo-dT beads (Invitrogen) and then fragmented using Ambion Fragmentation buffer using incubation time of 1 min 50sec at 70C. Fragmented RNA was then cleaned-up using 2.5X volume on SPRI beads (Agencourt), PNK treated (T4 PNK, NEB) and incubated with Terminator Exonuclease (Epicentre) for 70 minutes. Reaction mixture was dephosphorylated with FastAP (Fermentas), cleaned (2.5X SPRI) and then lygated to a linker1 (5Phos/AXXXXXXXXAGATCGGAAGAGCGTCGTGTAG/3ddC/, XXXXXXXX is an internal barcode specific for each sample,) using T4 RNA ligase I (NEB). Ligated RNA was cleaned-up by Silane beads (Dynabeads MyOne, Life Technologies) and pooled into a single tube. RT was then performed for the pooled sample, with a specific primer (5´-CCTACACGACGCTCTTCC-3´) using AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies). Then, RNA-DNA hybrids were degraded by incubating the RT mixture with 10% 1M NaOH (e.g. 2ul to 20ul of RT mixture) at 70C for 12 minutes. pH was then normalized by addition of corresponding amount of 0.5M AcOH (e.g. 4ul for 22 ul of NaOH+RT mixture). The reaction mixture was cleaned up using Silane beads and second lygation was performed, where 3’end of cDNA was lygated to linker2 (5Phos/AGATCGGAAGAGCACACGTCTG/3ddC/) using T4 RNA ligase I. The sequences of linker1 and linker2 are partially complementary to the standard Illumina read1 and read2/barcode adapters, respectively. Reaction Mixture was cleaned up (Silane beads) and PCR enrichment was set up using enrichment primers 1 and 2 (5’- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’, 5’-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGAC  GTGTGCTCTTCCGATCT-3’, where XXXXXXX is barcode sequence) and Phusion HF MasterMix (NEB). 10-12 cycles of enrichment were performed depending on the initial input amount of RNA. After clean-up with 0.8X volume of SPRI beads library was ready for characterization by Bioanalyzer.
3’ RNase H-sequencing: 50ng-1ug of total RNA was fragmented using NEBNext magnesium RNA fragmentation module (NEB) with incubation time of 2 minutes at 94C. Fragmented RNA was cleaned-up using 2.5X volume on SPRI beads (Agencourt), and then subjected to PolyA selection using Oligo-dT beads (Invitrogen). PolyA+ RNA was incubated with oligoDT primer (Fermentas) and polyA tails were digested by RNAseH (NEB) treatment for 1 hour at 37C. RNA was then cleaned (Silane beads), dephosphorylated with FastAP (Fermentas), cleaned (Silane beads) and lygated to a linker1 using T4 RNA ligase I (NEB). Ligated RNA was cleaned-up by Silane beads (Dynabeads MyOne, Life Technologies) and pooled into a single tube. Library preparation was then processed in the same way as in ExoCAGE protocol starting with RT step and so on.
3’ RNA-Seq without RNase H treatment: 50ng-1ug of total RNA was fragmented using NEBNext magnesium RNA fragmentation module (NEB) with incubation time of 5 minutes at 94C. Fragmented RNA was cleaned-up using 2.5X volume on SPRI beads (Agencourt), dephosphorylated with FastAP (Fermentas), cleaned (2.5X SPRI) and then ligated to a linker1 using T4 RNA ligase I (NEB). Ligated RNA was cleaned-up by Silane beads (Dynabeads MyOne, Life Technologies) and pooled into a single tube. The pooled sample was then subjected to poly(A) selection using Oligo(dT) beads (Invitrogen). Library preparation was then processed in the same way as in the ExoCAGE protocol starting with RT step and so on.


title: ExoCAGE_29C_zt15

description: processed data file: ExoCAGE.merged.wig

source_name: 5’ sequenced using ExoCAGe


title: RNAseH_29C_zt3

description: processed data file: RNsseH.merged.wig


title: YW_29C_1


title: RNAseH_18C_zt15

description: processed data file: RNsseH.merged.wig


title: RNAseH_18C_zt3

description: processed data file: RNsseH.merged.wig


title: CS2_H3K4me3 [ChIP-seq]


title: RNAseH_25C_zt15

description: processed data file: RNsseH.merged.wig


title: ExoCAGE_29C_zt3

description: processed data file: ExoCAGE.merged.wig


title: ExoCAGE_25C_zt23

description: processed data file: ExoCAGE.merged.wig


title: YW_18C_3


title: ExoCAGE_25C_zt9

description: processed data file: ExoCAGE.merged.wig


title: ExoCAGE_25C_zt3

description: processed data file: ExoCAGE.merged.wig


title: 3end_100bp_2


title: ExoCAGE_18C_zt23

description: processed data file: ExoCAGE.merged.wig


title: ExoCAGE_25C_zt15

description: processed data file: ExoCAGE.merged.wig


title: RNAseH_29C_zt15

description: processed data file: RNsseH.merged.wig


title: ExoCAGE_18C_zt9

description: processed data file: ExoCAGE.merged.wig


title: ExoCAGE_18C_zt3

description: processed data file: ExoCAGE.merged.wig


title: 3_29C_zt23

description: processed data file: 3_false.merged.tdf, 3_true.merged.tdf

source_name: 3’ RNA-Seq (without RNase H treatment)


title: 3_29C_zt19