title: SMN1 wildtype and mutant splicing

city: Irvine

first: Klemens

last: Hertel

state: CA

address: 19182 Jamboree Rd. 811 Med Sci I, C270

department: MMG

institute: UCI

data_processing:
Illumina Casava1.5 software was used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Reads were aligned to a custom index consisting of genomic SMN1 exons 6 to 8, spliced mRNA sequence consisting of exons 6, 7, and 8, and all exon 7 mutants that were introduced into the library. The reads were classified as either input reads, which would be the unprocessed DNA based reads, and output reads which would comprise all reads that were sequenced from processed mRNAs, which would include undergoing transcription and splicing.
Custom Python scripts to identify reads with wild-type exon 7 and all mutant exon 7 types.  Regular expression search functions were employed to search for multiple anchor sequences associated with exon 6, exon 7, exon 7 mutants, and exon 8. In order to determine the existence of aberrant splicing events we checked each read that contained these anchor sequences for either unexpected additional sequence inserted between exon anchor sequences or sequence deleted from the expected anchor sequences that result in partial anchor sequences.  Aberrant splicing event rates were calculated as percentages where the total number of normal reads divided by the total number of reads that contained each distinct event. Reads where anchor sequences for exon 6 and exon 8 were found, but no anchor sequences from exon 7 were considered to be non-canonically spliced reads where exon 7 was excised. Reads that did not contain anchor sequences or with multiple quality score based errors resulting in ambiguous nucleotides were discarded.
To calculate the effect of mutations on splicing fidelity, taking into account the rarity of events, we utilized odds ratios (OR) to determine those mutations that significantly change the ratios of aberrant splicing events compared to the aberrant splicing event rates observed in the wild-type SMN exon 7.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files that include non-canonical splicing event counts in wild type and mutant SMN1 exon6-exon8.

source_name: HeLa_SMN1 wildtype and mutant splicing

growth_protocol: HeLa cells used in this work were maintained at 37°C in monolayer in Dulbecco’s high glucose modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum, 4mM L-Glutamate, and 1mM Na-Pyruvate. Their cell confluence was kept to ~80% or less before splitting cells. Cells were transfected according to manufacturer’s specifications for Lipofectamine 2000 (Invitrogen) for plate sizes of 10cm, 6-well plates with 3cm wells, or 15cm plates.

extract_protocol:
Total RNA was purified from the cells using TRIzol reagent (Ambion) according to manufacturer’s recommendations and treated with Dnase.
RNA libraries were prepared for sequencing using standard Illumina protocols, except using SMN1 sequence specific primers to generate the sequenced library.

tag: cell line background

value: HeLa

value: transfected with a library of SMN1 mini-gene (exon6-exon7-exon8) constructs


title: 200923040024_R05C01_Buccal

status: Public on Sep 24 2019

type: genomic

city: Vancouver

first: Michael

middle: S.

last: Kobor

state: BC

address: 950 West 28th Avenue

department: Medical Genetics

country: Canada

institute: Centre for Molecular Medicine and Therapeutics / University of British Columbia

hyb_protocol: Bisulfite converted DNA was amplified, fragmented and hybridized to Illumina Infinium HumanMethylationEPIC Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged with BeadArray Reader using standard recommended Illumina scanner setting

data_processing: GenomeStudio software V2011.1 with GenomeStudio Methylation Analysis Module Version 1.9.1000 (background-subtraction, colour-correction)

label: Cy5 and Cy3

molecule: genomic DNA

organism: Homo sapiens

source_name: buccal swab genomic DNA

label_protocol: Standard Illumina Protocol

extract_protocol: Genomic DNA was extracted using the Gentra Puregene Buccal Cell Kit (Qiagen #158845) and DNA was bisulfite converted using the EZ DNA Methylation Kit™ (Zymo Research).

tag: age

tag: gender

value: M


title: WS_MSC_rep2

type: SRA

city: CUHK, Shatin

first: Hoi-Hung

last: Cheung

address: Rm.708A, LKS Integrated Biomedical Sciences Bldg.

department: School of Biomedical Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

library_source: transcriptomic

data_processing:
Raw reads are filtered to remove reads containing adapters or reads of low quality, so that downstream analyses are based on clean reads.
Algorithm for mapping sequences: appropriate software is chosen according to the characteristics of the reference genome. In general, TopHat2 is chosen for animal and plant genomes.
Files are provided in BAM format, a standard file format that contains mapping results, and the corresponding reference genome and gene annotation file for some species.
Gene expression level is measured by transcript abundance. The greater the abundance, the higher is the gene expression level. In our RNA-seq analysis, the gene expression level is estimated by counting the reads that map to genes or exons. Read count is not only proportional to the actual gene expression level, but is also proportional to the gene length and the sequencing depth. In order for the gene expression levels estimated from different genes and experiments to be comparable, the FPKM is used. In RNA-seq, FPKM, short for the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairssequenced, is the most commonest method of estimating gene expression levels, which takes into account the effects of both sequencing depth and gene length oncounting of fragments(Trapnell, Cole, et al., 2010).
To compare gene expression levels under different conditions, an FPKM distribution diagram and violin Plot are used. For biological replicates, the final FPKM would be the mean value.
Genome_build: hg19
Supplementary_files_format_and_content: Excel file includes FPKM values for all samples

molecule: total RNA

source_name: MSC

growth_protocol: MSC was grown in MSC medium (10% MSC-qualified FBS in low-glucose DMEM) and passed every 4-5 days when cells became confluent.

extract_protocol:
Trizol reagent
After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment.

treatment_protocol: No specific treatment.

tag: cell type

value: iPSC-derived

tag: genotype

value: WRN-null


title: C21_MSC_rep1

value: WRN+/+


title: 7539308067_R04C01

data_processing:
GenomeStudio software V2011.1 with GenomeStudio Methylation Analysis Module Version 1.9.1000 (background-subtraction, colour-correction)
Average Beta (after background-subtraction and colour-correction in GenomeStudio software according to standard Illumina protocol)
Unmethylated and methylated signal intensities

extract_protocol: Buccal swabs were collected using Catch-All Swabs (Epicentre, USA).  Genomic DNA was extracted using the Masterpure system (Epicentre, USA), and DNA was bisulfite converted using the EZ DNA Methylation Kit™ (Zymo Research).


title: 7539308067_R04C02


title: 200923040024_R03C01_Buccal12

value: F


title: 200923040024_R07C01_Buccal12


title: 200923040024_R01C01_Buccal


title: 200923040042_R04C01_Buccal


title: Tumor_Lungs_Thy1.1_Par_Thy1.1_7-1

city: Boston

first: Kornelia

last: Polyak

state: MA

address: 450 Brookline Ave

department: Medical Oncology

country: USA

institute: Dana-Farber Cancer Institute

data_processing:
RNA-Seq: Basecalls were performed using CASAVA version 1.8.2
RNA-Seq: Quality check of the called bases of the reads were performed using the FastQC tool (Babraham Bioinformatics). Tumor sample fastq files were trimmed to 40 bases.
RNA-Seq: Datasets of tumor origin were aligned to the human reference GRCh37/hg19 genome and datasets of stroma origin were aligned to the mouse reference GRCm38/mm10 genome, both using the STAR RNA-Seq aligner (version STAR_2.5.1b). Two-pass mapping was performed using the following modified parameters: --outSAMstrandField intronMotif, --outFilterMultimapNmax 20, --alignSJoverhangMin 8, --alignSJDBoverhangMin 1, --outFilterMismatchNmax 999, --outFilterMismatchNoverLmax 0.1, --alignIntronMin 20, --alignIntronMax 1000000, --alignMatesGapMax 1000000, --outFilterType BySJout, --outFilterScoreMinOverLread 0.33, --outFilterMatchNminOverLread 0.33, --limitSjdbInsertNsj 1200000, --chimSegmentMin 15, --chimJunctionOverhangMin 15, --twopassMode Basic. Samples were filtered to remove contamination (mouse contamination in tumor samples and human contamination in stroma samples). Reads uniquely mapped only to the target genome were kept, along with uniquely mapped reads that had significantly better alignment scores in the target genome compared to the contamination genome.
RNA-Seq: The read counts for individual genes were generated using the htseq-count script of the HTSeq framework (version 0.6.1p1) using modified parameters (--stranded no) and the hg19/mm10 refGene annotation file available at the UCSC Genome Browser.
RNA-Seq: For stroma samples, libraries were ran twice and reads for these duplicates were added together in the final gene-level counts file (Janiszewska_stroma-gene-level-counts.csv).
scRNA-seq: Samples were demultiplexed to FASTQ files using CellRanger v. 1.2. with cellranger mkfastq
scRNA-seq: FASTQ files were aligned and filtered using CellRanger v. 1.2. with cellranger count
scRNA-seq: Samples were aggregated into a single combined matrix and sequencing depth was normalized across runs, using CellRanger v. 1.2. with cellranger aggr
scRNA-seq: Data was filtered by selecting all genes expressed in at least 1 single cell, and all cells with at least 100 and at most 5,000 expressed genes, and with a maximum percent of mitochondrial DNA of 0.1 using the Seurat R package, v. 2.1.0. This amounted to keeping a total of 18,203 genes and 7,704 cells.
Genome_build: RNA-Seq and scRNA-Seq: hg19/mm10
Supplementary_files_format_and_content: RNA-Seq: Tab-separated file of the raw read counts of each samples for each gene.
Supplementary_files_format_and_content: scRNA-seq: Tab-separated file of the UMI counts generated by 10x CellRanger and filtered with Seurat

source_name: tumor

extract_protocol:
The mRNA selection and whole transcriptome amplification were performed as previously described with some modifications. A primer 5’-phosphorylated oligo-dT24 (pdT24) was used to select mRNA from total RNA. For denaturation and primer annealing, total RNA was mixed with dNTPs (25 mM) and pdT24, and put in a preheated 68°C PCR machine for 5 min and immediately placed on ice. Reverse transcription was performed with the addition of the First-Strand Buffer, DTT, RNase Inhibitor, and 10 U/μl Superscript Reverse Transcriptase III. The double-strand cDNA was generated by the addition of 30 μl mix containing 10x second100 strand buffer, dNTPs (25 mM), E.coli DNA ligase (10 U/μl), RNaseH, and DNA Polymerase (10U/μl), followed by 3 hr incubation at 16°C and 5 min at 70°C. The product was purified with the Genomic DNA Clean and Concentrator kit (Zymo). Then, DNA end-bluting, 5’-end phosphorylation, and ligation were performed with End-It DNA End-Repair Kit (Epicentre) and T4 DNA ligase (Epicentre). The product was directly amplified by adding the mixture of the Reaction Buffer and DNA polymerase from REPLI-g UltraFast Kit (Qiagen) in 15:1 ratio. The amplification reaction was carried at 30°C for 2 hr. The amplified product was purified using the same Genomic DNA Clean and Concentrator kit (Zymo). When eluted in 50 μl elution buffer, typically 5-10 μg of mRNA-derived cDNA amplicons were obtained. The amplicons were then evaluated for quantity, coverage, and purity by PCR using a set of primers for housekeeping genes (GAPDH, B2M), cDNA 5’ end (CREB5), and gDNA (10p).
cDNA libraries were constructed using Nextera XT DNA Sample preparation kit (Illumina) according to the manufacturer’s recommendations with minor modifications. Briefly, cDNA amplicons were fragmented and barcoded by tagmentation using Tn5 DNA transposase. Tagmented cDNA were amplified with a limited-cycle PCR program. During the PCR step, indices 1 and 2 were added to allow for cluster generation and multiplexed sequencing. The extension time was increased from 30s to 60s. The amplified cDNA libraries were purified and cleaned using 0.6x Agencourt AMPure XP (Beckman Coulter) and fresh ethanol, and eluted in TE buffer. The quality of sequencing libraries was assessed using a high-sensitivity DNA chip (Agilent). Finally, the libraries were sequenced deeply using the HiSeq2000 sequencer (Illumina). For the single cell RNA-Seq data, the cells were processed according to the 10xGenomics sample preparation protocol (Chromium Single Cell 3’ v2 Reagent Kit, 10xGenomics).

tag: clonality

value: monoclonal

tag: site

value: metastasis


title: Tumor_Lungs_Thy1.1_Par_Thy1.1_3-1


title: 7539308067_R03C01


title: Tumor_Lungs_CFP_Par_CFP_8-1

value: CFP


title: Tumor_Lungs_CFP_Par_CFP_4-3


title: 7539308067_R02C02


title: Tumor_Lungs_IL11_IL11_6-2


title: 200923040008_R08C01_Buccal


title: 200923040008_R05C01_Buccal12


title: Tumor_Lungs_IL11_IL11_6-1


title: Tumor_Lungs_IL11_IL11_10-2


title: 200923040008_R04C01_Buccal12


title: Tumor_Lungs_IL11_IL11_10-1


title: 7539308067_R01C02


title: 7539308067_R02C01


title: Tumor_Lungs_FIGF_FIGF_5-4

value: FIGF


title: Tumor_Lungs_FIGF_FIGF_5-3


title: 7539308067_R01C01


title: 7539308067_R03C02


title: 200923040008_R02C01_Buccal


title: WS_MSC_rep1


title: Tumor_Lungs_FIGF_FIGF_9-1


title: 7539308061_R06C02


title: Tumor_Lungs_FIGF_FIGF_9-3


title: 200925700093_R06C01_Buccal


title: Tumor_Lungs_Mixed_Mixed_1-1

value: polyclonal

value: mixed


title: Tumor_Lungs_Mixed_Mixed_1-4


title: 200925700093_R05C01_Buccal


title: Tumor_Primary_CFP_Par_CFP_4-3

value: primary tumor


title: Tumor_Primary_Par_mch_Par_mch_11-2

value: parental


title: 200923040008_R03C01_Buccal


title: 7539308061_R06C01


title: Tumor_Primary_Thy1.1_Par_Thy1.1_3-1


title: 200925700093_R08C01_Buccal12


title: 7539308061_R05C01


title: iBC_MSC_rep1


title: Tumor_Primary_IL11_IL11_10-2


title: Tumor_Primary_IL11_IL11_10-1


title: 7539308061_R04C02


title: Tumor_Primary_FIGF_FIGF_9-3


title: Tumor_Primary_FIGF_FIGF_9-1


title: Tumor_Primary_Mixed_Par_CFP_1-4


title: 7539308061_R04C01


title: Tumor_Primary_Mixed_IL11_1-4


title: 200925700089_R08C01_Buccal


title: 200925700089_R04C01_Buccal12


title: Tumor_Primary_CFP_Par_CFP_4-4


title: Tumor_Primary_Mixed_Par_Thy1.1_1-4


title: Tumor_Primary_Mixed_IL11_1-1


title: 200925700089_R05C01_Buccal12


title: 7539308061_R02C02


title: Tumor_Primary_Mixed_Par_Thy1.1_1-1


title: 7539308061_R05C02


title: Tumor_Primary_Mixed_FIGF_1-4


title: 7539308061_R03C01


title: Tumor_Primary_Mixed_FIGF_1-3


title: Tumor_Primary_Mixed_IL11_1-3


title: 200925700089_R01C01_Buccal12


title: 7539308061_R03C02


title: 7539308061_R02C01


title: sample_17_L002

city: Toronto

first: Igor

last: Jurisica

state: Ontario

address: 60 Leonard Avenue

department: Krembil Research Institute

institute: University Health Network

description: Replicate 2

data_processing:
Raw RNA sequencing data were preprocessed and normalized as follows:
RNAseq data were aligned against hg38 reference genome, using bowtie2.
Reads counts per gene were obtained using R packages GenomicRange and GenomicFeatures and using UCSC hg38 KnowGene database as a reference for genomic locations (TxDb.Hsapiens.UCSC.hg38)
Counts were normalized to counts per million.
Additional quantile normalization was applied.
Genome_build: HG38 (TxDb.Hsapiens.UCSC.hg38)
Supplementary_files_format_and_content: Entrez ID, Gene Symbol, Counts per millions as obtained across samples and their replicates

source_name: Lung to brain metastasis cells

growth_protocol: Brain metastases (BM) originating from non-small cell lung carcinoma (NSCLC) primary samples were processed and maintained in Neurocult media (STEMcell Technolgies) as brain metastasis initiating cells (BMICs). BMICs were grown as tumorspheres that were maintained at 37°C with a humidified atmosphere of 5% CO2

extract_protocol:
RNA was extracted from BMICs using Norgen total RNA kit.
Library prep was performed using the NEBNext Ultra Directional RNA Library Prep Kit along with the NEBNext Poly(A) mRNA Magnetic Isolation Module. Libraries were subject to further BioAnalyzer QC and quantified by qPCR.

tag: bmic line

tag: stage of the metastatic cascade

value: BMIC

tag: biological sample id

value: Sample 17


title: 200925700093_R03C01_Buccal12


title: sample_18_L001

description: Replicate 1

value: BMIT

value: Sample 18


title: 7539308061_R01C01


title: 200925700086_R06C01_Buccal

value: M


title: sample_18_L002

description: Replicate 2


title: 200925700086_R08C01_Buccal


title: sample_17_L001

description: Replicate 1


title: 200925700089_R03C01_Buccal_REP1


title: Tumor_Primary_Mixed_FIGF_1-1


title: 7539308061_R01C02


title: sample_16_L001

description: Replicate 1

value: Sample 16


title: 3998568023_R06C02


title: 200925700086_R04C01_Buccal12


title: 3998568023_R04C01


title: 200925700086_R05C01_Buccal


title: 200925700086_R01C01_Buccal12


title: sample_15_L002

description: Replicate 2

value: Sample 15


title: 200925700071_R07C01_Buccal12


title: 3998568023_R03C02


title: sample_13_L002

description: Replicate 2

value: LT

value: Sample 13


title: sample_12_L002

description: Replicate 2

value: Sample 12


title: sample_13_L001

description: Replicate 1


title: sample_14_L001

description: Replicate 1

value: Sample 14


title: sample_11_L002

description: Replicate 2

value: Sample 11


title: 200925700086_R03C01_Buccal12


title: sample_15_L001

description: Replicate 1


title: 200925700071_R06C01_Buccal


title: sample_10_L002

description: Replicate 2

value: Sample 10


title: sample_11_L001

description: Replicate 1


title: 3998568004_R03C02


title: 3998568023_R01C02

value: F


title: sample_10_L001

description: Replicate 1


title: sample_14_L002

description: Replicate 2


title: 3998568004_R03C01


title: 3998568023_R06C01


title: 200925700071_R05C01_Buccal


title: sample_12_L001

description: Replicate 1


title: sample_9_L002

description: Replicate 2

value: Sample 9


title: sample_9_L001

description: Replicate 1


title: 3998568004_R02C02


title: 3998567077_R06C02


title: 200925700071_R02C01_Buccal12


title: sample_8_L001

description: Replicate 1

value: Sample 8


title: 200925700071_R04C01_Buccal


title: sample_7_L002

description: Replicate 2

value: Sample 7


title: 3998567077_R06C01


title: 3998567077_R03C02


title: sample_16_L002

description: Replicate 2

value: BMIC


title: sample_8_L002

description: Replicate 2


title: sample_5_L002

description: Replicate 2

value: BT

value: Sample 5


title: sample_6_L002

description: Replicate 2

value: Sample 6


title: 200923040055_R05C01_Buccal

value: M


title: 200923040055_R07C01_Buccal12


title: 3998567077_R03C01


title: sample_6_L001

description: Replicate 1


title: sample_5_L001

description: Replicate 1


title: 3998567077_R02C02


title: sample_4_L001

description: Replicate 1

value: Sample 4


title: sample_4_L002

description: Replicate 2


title: sample_3_L002

description: Replicate 2

value: Sample 3


title: 200923040055_R02C01_Buccal


title: sample_3_L001

description: Replicate 1


title: sample_2_L002

description: Replicate 2

value: original

value: Sample 2


title: 3998567024_R06C01


title: 200923040055_R01C01_Buccal


title: sample_2_L001

description: Replicate 1


title: 3998567024_R05C02


title: sample_1_L002

description: Replicate 2

value: Sample 1


title: sample_7_L001

description: Replicate 1

value: BMIT


title: 200925700053_R07C01_Buccal


title: 3998567024_R03C02


title: 200925700053_R08C01_Buccal


title: 3998567024_R05C01


title: 3998567024_R02C02


title: sample_1_L001

description: Replicate 1


title: 200925700053_R04C01_Buccal12


title: 200925700046_R08C01_Buccal12


title: 200925700053_R02C01_Buccal12


title: 3998567024_R01C01


title: 200923040055_R04C01_Buccal12


title: 200925700053_R06C01_Buccal


title: 3998567024_R02C01


title: 200925700046_R04C01_Buccal


title: 200925700046_R07C01_Buccal


title: 100946150149_R01C01


title: 200925700046_R03C01_Buccal


title: 200925700046_R02C01_Buccal12


title: 100946150122_R06C02


title: 100946150122_R05C02


title: DSS miR-494 KO

city: Shanghai

first: Lixing

last: Zhan

address: 320 Yueyang Road

country: China

institute: Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

description:
total RNA  is the mixture from two independent samples
organoid from 3%DSS treated miR-494 KO mice_1
organoid from 3%DSS treated miR-494 KO mice_2

data_processing:
Illumina  Hiseq 2500 software used for basecalling.
the pair-ended reads were mapped to the mouse genome (UCSC mm10) using STAR v2.7 with NCBI RefSeq genes as the reference.
Further analyses were performed using R v3.3, including packages ggplot2, gplots, Rtsne, gdata, gsva, prcomp, and GOstats were used.
Genome_build: mm10
Supplementary_files_format_and_content: Excel files include FPKM values and count for each Sample

organism: Mus musculus

source_name: colonic crypts

extract_protocol:
Following euthanasia of DSS-treated mice, the entire colon was removed, everted, and rinsed 5 times with cold PBS (supplemented with 2% (vol/vol) penicillin and streptomycin). The rinsed colon was immersed into chilled (0-4 ºC) cell recovery solution buffer (354253, Corning, NY, USA) for 30-40 min; the resulting cell suspension was filtered through 70-μm strainers and rinsed with basic cell culture medium (DMEM/F12 (GibcoTM, Waltham, MA) supplemented with 1% (vol/vol) penicillin and streptomycin (GibcoTM), 1% glutamax (Gibco GibcoTM), and 1% HEPES (GibcoTM)) until the supernatant remained clear after centrifugation at 200xg for 2-3 min. RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: WT and miR-494-/- mice were provided with free access to 3% DSS (160110, MW=36-50 kD; MP Biomedicals, Santa Ana, CA, USA) in the drinking water for 7 days; the first day of DSS access was designated Day 0).

value: colonic crypts


title: DSS miR-494 WT

description:
total RNA  is the mixture from two independent samples
organoid from 3%DSS treated miR-494 WT mice_1
organoid from 3%DSS treated miR-494 WTmice_2


title: 200925700046_R01C01_Buccal12


title: poly_shERa_rep4

city: Solna

first: Ola

last: Larsson

address: Tomtebodavägen 23A

country: Sweden

institute: Karolinska Institute

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402695)

data_processing:
RNAseq reads were preprocessed using the nf-core/rnaseq v1.3 pipeline available at https://github.com/nf-core/rnaseq. HISAT2 was selected as alignment tool and all other parameters were kept as default.
Genes with multiple loci were excluded from the analysis. The analysis was performed on protein-coding genes
Genome_build: GRCm38
Supplementary_files_format_and_content: Raw read counts are provided for all samples in a tab delimited file

molecule: other

source_name: PTEN-deficient prostate cancer cell line (BM67) derived from the PB-Cre;PtenFlox/Flox mouse model of prostate cancer

growth_protocol: cells were grown in RPMI-1640 (Gibco) and supplemented to a final concentration of 5% Fetal Bovine Serum (FBS, Sigma) and 100 IU/mL penicillin and 10 µg/mL streptomycin (P/S, Invitrogen) and kept in a humidified incubator at 37°C supplemented with 5% CO2.

extract_protocol:
Polysome profiling was performed on four replicates of each condition as previously described (Gandin et al. J Vis Exp JoVE, 2014). Briefly, cytosolic lysates were loaded on a 5-50% sucrose gradient allowing for isolation of mRNAs associated with more than 3 ribosomes (hereafter referred to as polysome-associated mRNA) after ultracentrifugation. Total cytosolic RNA was isolated in parallel (Gandin et al. J Vis Exp JoVE, 2014).
RNAseq libraries were prepared using the Smart-seq2 method as described previously (Picelli et al, 2014), using 10ng RNA as input material. After pre-amplification PCR, 80pg of cDNA was used for tagmentation using the Nextera XT Kit (Illumina) in a total volume of 25uL.

treatment_protocol: Stable knockdown of ERα was achieved using the pGIPZ vector (Open Biosystems) containing a non-silencing control (shCtrl) or a mouse ERα shRNA (shERa)

tag: tissue

tag: rna type

value: Polysome-associated RNA

tag: shRNA

value: shERa


title: 100946150122_R03C02

value: F


title: C21_MSC_rep2

value: iPSC-derived

value: WRN+/+


title: 200925700027_R08C01_Buccal


title: poly_shERa_rep3

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402696)

value: PTEN-deficient prostate cancer cell line (BM67)


title: cyto_shERa_rep3

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402690)

value: Cytosolic RNA


title: poly_shERa_rep2


title: 100946150149_R02C02


title: 200925700093_R01C01_Buccal


title: 100946150149_R03C01


title: 200925700027_R06C01_Buccal12


title: 100946150121_R06C02


title: poly_shCtrl_rep4

value: shCtrl


title: 100946150121_R04C02


title: cyto_shCtrl_rep4


title: cyto_shERa_rep1

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402688)


title: cyto_shERa_rep2


title: 200925700027_R05C01_Buccal


title: 100946150121_R03C02


title: 200925700027_R03C01_Buccal


title: cyto_shCtrl_rep3

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402687)


title: 100946150122_R04C01


title: 100946150121_R01C02


title: 200925700025_R08C01_Buccal


title: cyto_shCtrl_rep2

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402686)


title: 100946150108_R06C02


title: 200925700025_R06C01_Buccal12


title: 200925700025_R05C01_Buccal12


title: poly_shERa_rep1

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402694)


title: cyto_shERa_rep4

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402689)


title: 100946150108_R04C01


title: shERa_2 nanoCAGE

description: Librairies were prepared with 2 barcodes (ACAGAT and GTATGA) for each sample and pooled. The pool was sequenced on 2 lanes. Reads from individual barcodes were extracted using TagDust (Lassmann,  BMC Bioinformatics, 2015) and each raw file provided here contains reads from one barcode and index sequenced on one lane.

data_processing:
Reads were trimmed to remove Nextera XT N-Series 3’ adaptors using cutadapt version 1.9.1 (Martin, Bioinforma Action, 2012) with the maximum read-adapter mapping error rate set to 0.15, the minimum overlap length set to 1 and trimming of up to 4 adaptors  was allowed. Remaining reads shorter than 25 bases were excluded.
Duplicated reads with identical UMI (the UMI identifier of each read can be found on the sequence identifier row after "FP:") and sequence (in the first 25 bp) were assumed to be PCR duplicates and only one was kept for further analysis.
A first alignment to the mouse genome mm10 was performed using Bowtie (version 0.12.7) (Langmead et al. Genome Biol, 2009) allowing no more than 2 mismatches in the first 28 bp on the 5’ end of the read. Reads which aligned uniquely (selected with parameters -m 1 –best –strata) and reads which did not align underwent a second alignment to a custom index of 5’ UTR sequences. For this index, refSeq 5’UTR sequences (O’Leary et al. Nucleic Acids Res, 2016) were extended with 78 bases upstream genomic and downstream mRNA sequences (78 is the maximum length of nanoCAGE reads after trimming of all adaptors). All best strata alignments with a maximum of 3 mismatches in the first 25 bases of the read were reported.
One or two bases were trimmed from the 5’ end of each read when alignment mismatches were present at the first or second position, respectively.
Strand invasion artefacts were removed using the perl script provided in Tang et al. (Nucleic Acids Res, 2013) with minimum edit distance set to 2.
Transcription start site peaks (defined as 5’ end mapping positions of reads) observed in less than 4 out of 6 samples (3 replicates and 2 barcodes per replicate) were excluded. Reads from all replicates were then merged. RefSeqs with less than 10 reads were excluded.
For each gene entrez identifier, the RefSeq(s) with most reads was (were) selected (for multiple RefSeqs with the same maximum count, all RefSeqs are kept).
The weighted 5’ UTR length was computed as a mean length along the RefSeq weighted by the read count of each observed peak.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited file with the following columns: entrez gene identifier (entrezid); RefSeq 5'UTR identifier to which the reads mapped (refSeq); total number of reads mapped to the corresponding RefSeq 5'UTR (nr_mapped_reads). weighted 5'UTR length (weighted_5UTR_length); 5'UTR length of sequence suggested by majority of reads (highest_peak_5UTR_length); sequence of 5'UTR with majority of reads (5UTRseq).

growth_protocol: shERα BM67 cells were grown until 80% confluency in RPMI-1640 (Gibco) and supplemented to a final concentration of 5% Fetal Bovine Serum (FBS, Sigma) and 100 IU/mL penicillin and 10 µg/mL streptomycin (P/S, Invitrogen) and kept in a humidified incubator at 37°C supplemented with 5% CO2

extract_protocol:
RNA was extracted with TRI-reagent (Sigma-Aldrich, Stockholm, Sweden) and further purified using the RNeasy MinElute Cleanup kit (Qiagen, Sollentuna, Sweden)
nanoCAGE libraries were prepared as described previously (Gandin et al. Genome Res, 2016) with several modifications detailed in the citation attached to this repository

treatment_protocol: Stable knockdown of ERα was achieved using the pGIPZ vector (Open Biosystems) containing a mouse ERα shRNA (shERa)


title: shERa_3 nanoCAGE


title: 100946150108_R03C01


title: shERa_2

description: BM67 shERα cells, small RNA rep 2

data_processing:
1: Read 3' trimming was performed using fastx_clipper (part of FASTX Toolkit version 0.0.14).
2: Reads which were less than 17nt-long were excluded. After this step, for miRNA quantification, data processing steps 3 to 6 were performed whereas for tRNA quantification steps 7 to 9 were performed instead.
3 (miRNA): Reads longer than 23 nt were also filtered out.
4 (miRNA): Remaining reads were aligned to the mm10 mouse genome using Bowtie version 1.0.1 (Langmead et al. Genome Biol, 2009) allowing one mismatch anywhere in the sequence. All alignments falling in the best alignment stratum were reported (parameters -v 1 –best –strata -a).
5 (miRNA): miRNA levels were quantified using the countOverlaps function of the GenomicAlignments R/Bioconductor package (Lawrence et al. PLoS Comput Biol, 2013) and the miRBase definition of miRNA coordinates Release 22 (Kozomara and Griffiths-Jones Nucleic Acids Res, 2014).
6 (miRNA): For miRNAs mapping to multiple genomic locations, the quantification with minimum total count was considered. Only mature miRNA counts were kept in the analysis.
7 (tRNA): the tRNAscan-SE algorithm was used to predict the presence of tRNA loci in the mm10 genome. These tRNA loci were “in-silico processed” (introns are removed, CCA is added, 5-prime G base is added to Histidine tRNAs, N-bases are added to the ends of each mature sequence) using the maketrnadb script. All tRNA loci and mature tRNAs were added to the mm10 genome and a Bowtie2 index was created
8 (tRNA): The default Bowtie2 alignment strategy was used (i.e. search for multiple alignments, report the best one, with upper limits for parameters –D and –R set at 100 (Langmead and Salzberg, Nat Methods, 2012)).
9 (tRNA): Quantification of tRNA as well as non coding RNA features (for normalization purposes (The RNAcentral Consortium, Nucleic Acids Res, 2017)) was performed using the ARM-seq (Cozen et al. Nat Methods, 2015) countreads.py script. All reads mapping tRNAs were retained, regardless of where in the tRNA they mapped.
Genome_build: mm10
Supplementary_files_format_and_content: For miRNA quantification: raw read counts for mature miRNAs are provided for all samples in a tab delimited file
Supplementary_files_format_and_content: For tRNA quantification: raw read counts for tRNAs and small non coding RNAs are provided for all samples in a tab delimited file

extract_protocol:
RNA was extracted in triplicates from ERα shRNA and control BM67 cells using the RNeasy Plus Mini Kit (Qiagen)
RNAseq libraries were prepared according to the Illumina TruSeq Small RNA Library Preparation protocol with small RNA enrichment on the Agilent Bravo Liquid Handling Platform


title: poly_shCtrl_rep3

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402693)


title: shERa_1

description: BM67 shERα cells, small RNA rep 1


title: 100946150108_R01C01


title: shERa_3

description: BM67 shERα cells, small RNA rep 3


title: 200925700025_R01C01_Buccal


title: 200925700019_R08C01_Buccal12


title: poly_shCtrl_rep2

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402692)


title: shCtrl_3

description: BM67 control cells, small RNA rep 3


title: shCtrl_1

description: BM67 control cells, small RNA rep 1


title: shCtrl_2

description: BM67 control cells, small RNA rep 2


title: 100946150107_R02C01


title: poly_shCtrl_rep1

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402691)


title: 100946150107_R01C02


title: shERa_1 nanoCAGE


title: 200925700019_R04C01_Buccal12


title: 200925700025_R03C01_Buccal


title: Sample_81790_FBTreg

city: Ann Arbor

first: Richard

middle: C

last: McEachin

state: United States

address: 2800 Plymouth Road

department: DCM&B

institute: University of Michigan

data_processing:
We downloaded the reads files from the Sequencing Core’s storage, and concatenated those into a single .fastq file for each sample.
We checked the quality of the raw reads data for each sample using FastQC (version 0.10.0) to identify features of the data that may indicate quality problems (e.g. low quality scores, over-represented sequences, inappropriate GC content, etc.).
We used the software package Tuxedo Suite for alignment, differential expression analysis, and post-analysis diagnostics. Briefly, we aligned reads from the samples from the two strains to respective E. Coli reference genome sequence (NCBI LF82, ENSEMBL K12 MG1655) [5, 6] using Bowtie2 (version 2.1.0). We used default parameter settings for alignment, with the exception of: “--b2-very-sensitive” telling the software to spend extra time searching for valid alignments.
We used FastQC for a second round of quality control (post-alignment), to ensure that only high quality data would be input to expression quantitation and differential expression analysis.
We used Cufflinks/CuffDiff (version 2.1.1) for expression quantitation and differential expression analysis, using respective reference genome sequence reference transcriptome annotation. For this analysis, we used parameter settings: “--multi-read-correct” to adjust expression calculations for reads that map in more than one locus, as well as “--compatible-hits-norm” and “--upper-quartile –norm” for normalization of expression values. We generated diagnostic plots using the CummeRbund package.
Genome_build: mm10
Supplementary_files_format_and_content: text file of raw mapped read counts

source_name: FBTreg

extract_protocol:
Cell lysates of FACS sorted cells were homogenized using QIAshredder, then total RNA samples were isolated using RNeasy Plus Micro Kit (Qiagen), including an on-column DNase treatment using RNase-Free DNase Set (Qiagen) according to manufacturer’s instruction.
RNA concentration and quality was determined and samples with RNA Integrity Numbers (RINs) of 8 or greater were prepped for strand mRNA libraries by the University of Michigan Sequencing Core. Libraries were multiplexed (6 per lane) and sequenced using paired end 50 cycle reads on a HiSeq 4000 (Illumina).

treatment_protocol: mice received DT for 3 weeks after 8 weeks’ recovery from 1- day caerulein treatment; pancraes tissues were harvested and digested for single cell suspension; pancreatic epithelial cells or fibroblasts were freshly sorted by FACS.

tag: genotype/variation


title: Sample_81792_FBTreg


title: Sample_81791_FBTreg


title: Sample_81789_FBControl

source_name: FBControl

value: KC


title: 100946150106_R05C02


title: 200925700019_R02C01_Buccal

value: M


title: Sample_81787_FBControl


title: Sample_81788_FBControl


title: Sample_81786_EpithTreg

source_name: EpithTreg

value: pancreatic epithelial cells (DAPI-CD45-EpCAM+)


title: 100946150106_R04C01


title: 200925700019_R01C01_Buccal12


title: Sample_81785_EpithTreg


title: cyto_shCtrl_rep1

description: This RNA sample was also quantified using DNA-microarrays (accession nr GSM3402685)


title: 200923040096_R07C01_Buccal12


title: 200925700019_R05C01_Buccal


title: 100946150106_R03C01


title: 100946150106_R01C01


title: Sample_81784_EpithTreg


title: Sample_81782_EpithControl

source_name: EpithControl


title: 100946150108_R04C02


title: Sample_81781_EpithControl


title: 200923040096_R05C01_Buccal12


title: Lgr5+Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3

city: Mainz

first: Sergi

last: Sayols

address: Ackermannweg 4

country: Germany

institute: Institute of Molecular Biology, Mainz

data_processing:
Reads were demultiplexed and fastq files were generated with Illumina bcl2fastq-1.8.4
Fastq files were mapped with STAR_2.3.1z13_r470 onto the iGenomes mm9 reference
Secondary alignments were filtered with samtools 1.2
Counts for every gene were calculated with HTSeq-0.6.1p1 using the gene model provided with the iGenomes mm9 reference from 2013-03-06
Counts were normalized and modeled with DESeq2_1.14.1
Genome_build: mm9
Supplementary_files_format_and_content: tab separated file containing counts per gene

molecule: polyA RNA

source_name: EpCAM+ embryonic intestinal epithelial cells_E13.5

extract_protocol:
500 embryonic intestinal epithelial cells were isolated by FACS directly in 7ml of lysis buffer (Clontech) supplemented with 5% RNase inhibitor and stored at -80°C. For each replicate, RNA was isolated from pull of three embryos (E13.5) or single embryos (E15.5). PolyA+ mRNA was used for cDNA synthesis using SMARTer v3.0 kit (Clontech) according to manufacturer’s instructions. Amplification was performed for 15 cycles.
After cDNA fragmentation (Covaris), libraries were prepared using Ovation Ultralow Library System (NuGEN) according to manufacturer’s instructions.

tag: strain background

value: mixed C57BL/6 CD1

tag: developmenatl stage

value: small intestine

value: Intestinal epithelial cells


title: 100946150097_R03C02


title: Lgr5+Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: Lgr5+Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: 200923040096_R02C01_Buccal


title: 100946150097_R02C02


title: 200923040096_R01C01_Buccal


title: EpCAM+Lgr5-Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: 100946150106_R03C02


title: EpCAM+Lgr5-Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: EpCAM+Lgr5-Id2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: 200925700015_R08C01_Buccal


title: 100946150061_R06C02


title: Sample_81783_EpithControl


title: 100946150061_R06C01


title: 200925700015_R06C01_Buccal12


title: Lgr5+Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: Lgr5+Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: 200925700015_R05C01_Buccal12


title: Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: 100946150061_R01C01


title: 200925700015_R03C01_Buccal


title: 100946150061_R04C02


title: Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: 200923040096_R04C01_Buccal


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 3

source_name: EpCAM+ embryonic intestinal epithelial cells_E15.5 posterior part


title: 200925700027_R07C01_Buccal12


title: 9407210042_R03C02


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 1


title: 200925700015_R01C01_Buccal


title: 9407210042_R03C01


title: 200925700002_R08C01_Buccal


title: Lgr5+Id2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 3


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 1


title: 9407210038_R06C01


title: 9407210038_R06C02


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 2


title: 200925700002_R05C01_Buccal


title: Lgr5+Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 2


title: Lgr5+Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 3


title: 200925700002_R04C01_Buccal12


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 3


title: Lgr5+Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 1


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 2


title: 200925700002_R02C01_Buccal


title: 200925700002_R01C01_Buccal12


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 1


title: 9407210038_R04C01


title: 9407210021_R06C01


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_posterior part of wt gut_repl 2


title: 9407210035_R04C01


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 3

source_name: EpCAM+ embryonic intestinal epithelial cells_E15.5 anterior part


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 2


title: 200925700001_R08C01_Buccal


title: 9407210021_R05C01


title: Lgr5+Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 1


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 3


title: 200925700001_R07C01_Buccal


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 2


title: 9407201158_R05C01


title: 200925700001_R05C01_Buccal12


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 3


title: 9407201156_R06C02


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 1


title: Axin2:tdTomato+_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 2


title: Lgr5+Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: 200925700001_R02C01_Buccal12


title: 9407201155_R04C02


title: 200925700001_R01C01_Buccal12


title: EpCAM+Lgr5-Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: 9407201155_R02C02


title: EpCAM+Lgr5-Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: Lgr5+Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: Lgr5+Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: 200923040065_R06C01_Buccal


title: EpCAM+Lgr5-Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: Lgr5+Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: 200923040065_R05C01_Buccal12


title: 9407201146_R04C01


title: EpCAM+Lgr5-Axin2:tdTomato-_E15.5_intestinal epitheliua cells labelled at E9.5_anterior part of wt gut_repl 1


title: Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: 200923040065_R04C01_Buccal


title: 9407201146_R04C02


title: 200923040065_R02C01_Buccal12


title: 200925700001_R03C01_Buccal


title: Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: 9407201156_R03C01


title: 9407201143_R05C01


title: Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep3


title: Lgr5+Axin2:tdTomato+_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep1


title: 200923040065_R01C01_Buccal12


title: Lgr5+Axin2:tdTomato-_E13.5_intestinal epitheliua cells labelled at E9.5_wt_rep2


title: dcl1-5 mRNA-seq (biorep 2)

city: Wageningen

middle: D

last: Nodine

state: Gelderland

address: Radix West

department: Plant Sciences

country: Netherlands

institute: Wageningen University & Research

description: Paired-end 50 base read Smart-seq2 data from second biological replicate of dcl1-5 globular embryos

data_processing:
sRNA-seq: Cutadapt (Martin, 2011) was used to trim adapter sequences from sRNA-seq reads and 18–30-base sequences that contained an adapter were retained. The trimmed sequences were aligned to the Arabidopsis thaliana TAIR10 genome (Lamesch et al., 2012) with STAR (Dobin et al., 2013) requiring no mismatches and allowing ≤100 multiple end-to-end alignments. Resulting SAM files were then processed with the readmapIO.py script to re-assign multimappers with a “rich-get-richer” algorithm as previously described (Schon et al., 2018). Output bedFiles were sorted, condensed and normalized for total genome-matching reads. The BEDtools map function (Quinlan and Hall, 2010) was then used to quantify the number of reads mapping to the same strand and overlapping ≥80% of mature miRNAs as annotated in TAIR10 and miRBase21 (Kozomara et al., 2019).
nanoPARE: Analysis of nanoPARE data was as described (Schon et al., 2018) except that all capped features identified in the embryonic series were merged with those from published floral bud samples (Schon et al., 2018), and used to mask capped features from the transcript-level bedGraph files. Additionally, we used EndCut (Schon et al., 2018) to test for significant target sites for 164 miRNAs detected ≥1 RPM in at least one embryonic stage. nanoPARE libraries from all post-embryonic tissues were analyzed in an identical manner.
mRNA-seq: Transcriptome analyses were as described (Hofmann et al., 2019) except that TAIR10 transposable element gene models were also included in the Kallisto-based pseudo-alignments (Bray et al., 2016).
Genome_build: TAIR10
Supplementary_files_format_and_content: TSV files for sRNA-seq processed data files (SAMPLE.quant.on.miRNA_mature.tsv) have 19 columns: 1) name of mature miRNA, 2-10) number of various base-long reads normalized based on number of alignments to genome, 11-19) number of various base-long reads normalized based on the number of alignments to the genome and the total number of million genome-matching reads in the library (i.e. RPM; reads per million genome-mathcing reads).
Supplementary_files_format_and_content: TSV files for nanoPARE processed data files (SAMPLE.emb.miRNA.exp.1.detected.sites.tsv) were output from EndCut (Schon et al. 2018) and have the following 10 columns: 1) target.transcript, target transcript; 2) sRNA, small RNA/miRNA); 3) slice.site, nt coordinate of predicted miRNA-mediated slice site in target transcript; 4) Allen.score, Allen Score of predicted miRNA:target transcript duplex; 5) slice.site.rpm, reads per million transcriptome-mapping reads (after capmasking); 6) fold.change, slice.site.rpm divided by the maxium site rpm within 20 or 50-nt; 7) verifed, whether or not slice site had been verified based on 5' RACE (in flowers); 8) common.name, common name of target gene (if it exists); 9) interaction, miRNA:target interaction; 10) adj.p.val, Benjamini-Hochberg adjusted P-value of significance of detected cleavage site determined by EndCut.
Supplementary_files_format_and_content: TSV files for mRNA-seq processed data files (SAMPLE.abundance.tsv) were output from Kallisto (Bray et al. 2016) and have following four columns: 1) target_id, transcript isoform identification; 2) length, length of annotated transcript isoform; 3) eff_length, effective length of transcript isoform; 4) est_counts, estimated counts based on pseudoalignment; 5) tpm, transcripts per million

organism: Arabidopsis thaliana

source_name: Globular embryos

growth_protocol: Controlled growth chamber at 20˚C-22˚C with a 16-h light/8-h dark cycle. Plants were four weeks old when tissue was harvested.

extract_protocol:
TRIzol reagent (Thermo Fisher Scientific)
For Samples 1-33, sRNA-Seq libraries were generated as described in the associated article. For Samples 34-66, nanoPARE libraries were generated as described in the associated article. For Samples 67-69, mRNA-seq libaries were generated according to the Smart-seq2 protocol (Picelli et al. 2013. Nature Methods).
sRNA-seq for Samples 1-33; nanoPARE for Samples 34-66; mRNA-Seq for Samples 67-69

tag: accession


title: dcl1-5 mRNA-seq (biorep 1)

description: Paired-end 50 base read Smart-seq2 data from first biological replicate of dcl1-5 globular embryos


title: 9407201145_R04C01


title: 9407201143_R01C02


title: 200923040047_R08C01_Buccal12_REP1


title: xrn4-5 shoots nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of xrn4-5 shoots

source_name: Seedling shoots


title: xrn4-5 shoots nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of xrn4-5 shoots


title: 9407201141_R01C01


title: 200923040047_R06C01_Buccal


title: 9407201137_R04C02


title: xrn4-5 root nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of xrn4-5 roots

source_name: Seedling roots


title: xrn4-5 root nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of xrn4-5 roots


title: xrn4-5 root nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of xrn4-5 roots


title: 9407201137_R01C02


title: 200923040047_R03C01_Buccal12


title: 200923040047_R04C01_Buccal


title: Mature green nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of mature green embryos

source_name: Mature green embryos

value: wild type


title: dcl1-5 globular nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of dcl1-5 globular embryos


title: dcl1-5 globular nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of dcl1-5 globular embryos


title: Mature green nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of mature green embryos


title: dcl1-5 globular nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of dcl1-5 globular embryos


title: 9407201134_R06C01

source_name: buccal swab genomic DNA


title: Mature green nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of matuire green embryos


title: 200923040017_R08C01_Buccal


title: 200923040017_R05C01_Buccal12


title: 9407201099_R04C02


title: Bent cotyledon nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of bent cotyledon embryos

source_name: Bent cotyledon embryos


title: Bent cotyledon nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of bent cotyledon embryos


title: 9407201099_R02C02


title: 200923040047_R05C01_Buccal12


title: 9407201137_R02C01


title: 9407201135_R05C01


title: 200923040017_R03C01_Buccal


title: Late torpedo nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of late torpedo embryos

source_name: Late torpedo embryos


title: Late torpedo nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of late torpedo embryos


title: Bent cotyledon nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of bent cotyledon embryos


title: Late torpedo nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of late torpedo embryos


title: 9407201013_R02C02


title: Early torpedo nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of early torpedo embryos

source_name: Early torpedo embryos


title: 200923040017_R02C01_Buccal12


title: Early torpedo nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of early torpedo embryos


title: Early torpedo nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of early torpedo embryos


title: 9407201013_R02C01


title: Late heart nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of late heart embryos

source_name: Late heart embryos


title: Late heart nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of late heart embryos


title: 9388306122_R06C01


title: Early heart nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of early heart embryos

source_name: Early heart embryos


title: Late heart nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of late heart embryos


title: 200923040014_R06C01_Buccal12


title: 200923040017_R04C01_Buccal


title: 9388306122_R04C02


title: Early heart nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of early heart embryos


title: Early heart nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of early heart embryos


title: 200923040014_R04C01_Buccal12


title: 9388306122_R01C02


title: 200923040014_R03C01_Buccal12


title: 9407201099_R02C01


title: 9388306054_R04C02


title: Globular nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of globular embryos


title: Globular nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of globular embryos


title: Preglobular nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of preglobular embryos

source_name: Preglobular (8-cell/16-cell) embryos


title: Globular nanoPARE (biorep 3)

description: Single-end 50 base read nanoPARE data from third biological replicate of globular embryos


title: 200923040014_R08C01_Buccal


title: 9388306054_R02C02


title: 200923040014_R07C01_Buccal


title: Preglobular nanoPARE (biorep 2)

description: Single-end 50 base read nanoPARE data from second biological replicate of preglobular embryos


title: 9388306017_R06C01


title: 200923040013_R08C01_Buccal


title: Leaf sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of leaves

source_name: Rosette and cauline leaves


title: Preglobular nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of preglobular embryos


title: Floral bud sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of floral buds

source_name: Unopened floral buds


title: 9388306017_R01C02


title: 200923040013_R07C01_Buccal12


title: 0.5 ng sRNA-seq

description: Single-end 50 base read sRNA-seq data from 0.5 ng of total RNA isolated from bent cotyledon embryos


title: 9388306016_R05C02


title: 200923040013_R03C01_Buccal


title: 1 ng sRNA-seq

description: Single-end 50 base read sRNA-seq data from 1 ng of total RNA isolated from bent cotyledon embryos


title: 200923040013_R02C01_Buccal12


title: 5 ng sRNA-seq

description: Single-end 50 base read sRNA-seq data from 5 ng of total RNA isolated from bent cotyledon embryos


title: dcl1-5 globular sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of dcl1-5 globular embryos


title: 200923040013_R01C01_Buccal


title: xrn4-5 shoots nanoPARE (biorep 1)

description: Single-end 50 base read nanoPARE data from first biological replicate of xrn4-5 shoots


title: 50 ng sRNA-seq

description: Single-end 50 base read sRNA-seq data from 50 ng of total RNA isolated from bent cotyledon embryos


title: 200923040014_R02C01_Buccal12_REP1


title: dcl1-5 globular sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of dcl1-5 globular embryos


title: Mature green sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of mature green embryos


title: Mature green sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of mature green embryos


title: 8784233144_R01C02


title: 8784233144_R06C01


title: 200923040007_R06C01_Buccal


title: 9388306016_R02C02


title: Bent cotyledon sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of bent cotyledon embryos


title: 200923040007_R05C01_Buccal


title: 8622007201_R03C01


title: Bent cotyledon sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of bent cotyledon embryos


title: 494-IEC

description: Cells were transfected with miR-494-3p-mimic for 24 hours and then treated with LPS for 24 hours. Total RNA  is the mixture from three independent samples

data_processing:
Raw reads were subjected to quality control (QC) to determine if a resequencing step is needed.
After QC, raw reads were filtered into clean reads which will be aligned to the reference sequences.Filtering steps are as follows:1) Remove reads with adaptors;2) Remove reads in which unknown bases are more than 10%;3) Remove low quality reads (the percentage of low quality bases is over 50% in a read, we define the low quality base to be the base whose  sequencing quality  is no more than 5).
Use  Bowtie2  to map clean reads to reference gene and use  BWA to reference genome.
RSEM  was used to computed maximum likelihood abundance estimates. FPKM  method is used in calculated expression level.
Use NOISeq to screen differentially expressed genes between two groups.
Genome_build: hg19
Supplementary_files_format_and_content: Processed data were txt format and all gene FPKM of each sample were contained in each txt file.

source_name: intestinal epithelial cells

growth_protocol: The isolated intestinal cells were cultured in growth medium [DMEM/F12 supplemented with 1% (vol/vol) insulin mix, 2.5% (vol/vol) FBS, and 2% (vol/vol) penicillin and streptomycin] for 5-6 days.

extract_protocol:
RNA was harvested using Trizol reagent followed by RNeasy Mini kit (Qiagen) cleanup. Total RNA was subjected to Hiseq RNA-Seq, performed by BGI Tech Solutions Co., Ltd. Transcriptome reads from RNA-Seq experiments were mapped to the reference genome (hg19) by using BWA/Bowtie tool. Genes expression level is quantified by a software package called RSEM.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: The isolated intestinal cells were cultured in growth medium [DMEM/F12 supplemented with 1% (vol/vol) insulin mix, 2.5% (vol/vol) FBS, and 2% (vol/vol) penicillin and streptomycin] for 5-6 days, followed by transfection with control-mimice or miR-494-3p mimice for 24 hours. The transfected cells were treated with LPS for another 24 hours.

value: intestinal epithelial cells


title: Mature green sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of matuire green embryos


title: NC-IEC

description: Cells were transfected with control-mimic for 24 hours and then treated with LPS for 24 hours.Total RNA  is the mixture from three independent samples.


title: 8622007168_R04C02


title: Late torpedo sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of late torpedo embryos


title: 8622007168_R03C02


title: Bent cotyledon sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of bent cotyledon embryos


title: 8622007201_R02C02


title: 200923040007_R01C01_Buccal12


title: Late torpedo sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of late torpedo embryos


title: dcl1-5 globular sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of dcl1-5 globular embryos


title: 200923040007_R08C01_Buccal12


title: 8622007168_R02C02


title: Late torpedo sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of late torpedo embryos


title: 200923040004_R08C01_Buccal


title: 200923040004_R06C01_Buccal12


title: 200923040004_R07C01_Buccal12


title: 7539308084_R06C01


title: Early torpedo sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of early torpedo embryos


title: 7539308084_R03C02


title: 200923040007_R03C01_Buccal


title: 200923040004_R03C01_Buccal


title: DN_6

city: La Jolla

first: Klaus

last: Ley

address: 9420 Athena Cir

institute: La Jolla Institute for Immunology

data_processing:
Reads that passed Illumina filters were filtered for reads aligning to tRNA, rRNA, adapter sequences, and spike-in controls. The reads were then aligned to the mm10 reference genome and NCBI annotations using TopHat (v 1.4.1). DUST scores were calculated with PRINSEQ Lite (v 0.20.3) and low-complexity reads (DUST > 4) were removed from the BAM files. The alignment results were parsed via SAMtools to generate SAM files. Read counts to each genomic feature were obtained with the htseq-count program (v 0.7.1) using the “union” option.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited Raw Expression counts file obtained through HTSeq. Samples as Columns, genes as rows

source_name: Aorta

extract_protocol:
Aortas and carotid arteries from male and female Apoe-/- Cx3cr1GFP Cd11cYFP fed WD for 5 months were enzymatically digested. Single cell suspensions were stained with CD45 and 7AAD. Live, CD45+ cells were flow sorted into GFP+ YFP- (GFP), GFP+YFP+ (DP), GFP-YFP+(YFP), and GFP-YFP- (DN) groups using a FACSAria II directly into Trizol LS. RNA was extracted from the sorted GFP, DP, YFP, and DN cells using an RNeasy Mini Kit (Qiagen) following the standard protocol.
Total RNA was processed using SMART-seq v4 Ultra Low Input RNA Kit (Clonetech) following manufacturer’s instructions. cDNA was amplified using 11 cycles and eluted in 12 μL. 5 μL of resulting cDNA was processed using a NexteraXT kit (Illumina) following manufacturer’s instructions.

tag: cell subset

value: GFP-YFP-

value: Aorta

tag: strain

value: 7 months (5 months Western Diet)

value: atherosclerotic Apoe-/- Cx3cr1GFP Cd11cYFP


title: DN_1

tag: age


title: Early torpedo sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of early torpedo embryos


title: YFP_5

value: YFP+


title: YFP_6


title: 200923040004_R01C01_Buccal

tag: gender

value: F


title: 7539308078_R05C01


title: YFP_4

tag: genotype


title: Early torpedo sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of early torpedo embryos


title: 7539308078_R01C01


title: 200923040002_R08C01_Buccal


title: Late heart sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of late heart embryos


title: Late heart sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of late heart embryos


title: YFP_3


title: 6057841135_R06C02

data_processing:
GenomeStudio software V2011.1 with GenomeStudio Methylation Analysis Module Version 1.9.1000 (background-subtraction, colour-correction)
Average Beta (after background-subtraction and colour-correction in GenomeStudio software according to standard Illumina protocol)

extract_protocol: Buccal epithelial cells were collected with using Isohelix Buccal Swabs (Cell Projects, Harrietsham, Kent, England) and stabilized per manufacturer’s instructions for storage at room temperature. Genomic DNA was isolated using Isohelix Buccal DNA Isolation Kits (Cell Projects, Harrietsham, Kent, England), and then purified and concentrated using DNA Clean & Concentrator (Zymo Research, Irvine, CA)


title: 6057841135_R04C02


title: DN_3


title: YFP_1


title: 6057841135_R03C02


title: Late heart sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of late heart embryos


title: 200923040002_R07C01_Buccal


title: 6057841135_R06C01


title: DP_5

value: GFP+YFP+


title: DP_4


title: 200923040002_R06C01_Buccal12


title: Early heart sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of early heart embryos


title: 200923040002_R01C01_Buccal12


title: Early heart sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of early heart embryos


title: DP_1


title: Lung_E16.5_3

city: Copenhagen

first: Nicolai

last: Krogh

address: Blegdamsvej 3B

department: Department of Cellular and Molecular Medicine

country: Denmark

institute: University of Copenhagen

description: Whole cell RNA from tissue

data_processing:
Library strategy: RiboMeth-seq
Barcode separation using python script
Adaptor trimming using Cutadapt v. 2.0
Mapping to rRNA reference sequence using Bowtie2 v. 2.3.4.1
Counting read ends and calculating RiboMeth-seq scores using python scripts
Genome_build: Human rDNA (18S, 28S, 5.8S, 5S). The reference sequences are in Supplementary_Data_1.fasta
Supplementary_files_format_and_content: MS Excel file contains 5' and 3' read count and calculated RiboMeth-seq score at all positions in the rRNA sequence.

source_name: Lung

extract_protocol:
Tissues were homogenized and whole cell RNA was extracted using Tri-Reagent (Euromedex) according to the manufacturers instructions.
RiboMeth-seq: 5-10 ug of RNA was partially degraded by alkaline at denaturing temperatures. The size fraction 20-40 nt was purified on gels and linkers added using a system relying on a modified Arabidopsis tRNA ligase joining 2',3' cyclic phosphate and 5' phosphate ends. The library fragments were then sequenced on the Ion Proton platform. See Birkedal U, Christensen-Dalsgaard M, Krogh N, Sabarinathan R, Gorodkin J, Nielsen H. Profiling of ribose methylations in RNA by high-throughput sequencing. Angewandte Chemie. 2015;54(2):451-5 for detailed description

value: Lung

tag: developmental stage


title: Lung_E16.5_2

description: Whole cell RNA from tissue


title: 6057841135_R04C01


title: Globular sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of globular embryos


title: GFP_6

value: GFP+


title: GFP_5


title: Globular sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of globular embryos


title: Globular sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of globular embryos


title: 6057841135_R01C01


title: 200923040002_R05C01_Buccal


title: 6057841123_R06C02


title: GFP_4


title: Lung_adult_3

description: Whole cell RNA from tissue

value: adult


title: Early heart sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of early heart embryos


title: dcl1-5 mRNA-seq (biorep 3)

description: Paired-end 50 base read Smart-seq2 data from third biological replicate of dcl1-5 globular embryos


title: GFP_3


title: Lung_adult_2

description: Whole cell RNA from tissue


title: Preglobular sRNA-seq (biorep 3)

description: Single-end 50 base read sRNA-seq data from third biological replicate of preglobular embryos


title: 6057841123_R05C02


title: DP_2


title: Lung_adult_1

description: Whole cell RNA from tissue


title: Preglobular sRNA-seq (biorep 2)

description: Single-end 50 base read sRNA-seq data from second biological replicate of preglobular embryos


title: GFP_2


title: 6057841123_R04C02

value: M


title: 6057841135_R03C01


title: GFP_1


title: Lung_E16.5_1

description: Whole cell RNA from tissue


title: Liver_E16.5_2

description: Whole cell RNA from tissue

source_name: Liver

value: Liver


title: 6057841123_R02C02


title: Liver_E16.5_1

description: Whole cell RNA from tissue


title: lib13_pos_d31

first: Carlos

last: Villacorta-Martin

address: 670 Albany St

institute: Center for Regenerative Medicine at Boston University

description: cell_names_final.txt,gene_names_final.txt,11_09_killian_final.mtx.gz

data_processing:
Data was demultiplexed and mapped using the inDrops and bcbio-nextgen pipeline
Further analysis were done in Seurat, SPRING and CSHMM
Differential expression tests run in MAST
Genome_build: GRCh37
Supplementary_files_format_and_content: The processed data includes a count matrix (mtx sparse format), the list of gene names and the list of cell names

source_name: iPSC derived cells sorted on NKX2-1^GFP at the lung primordial progenitor stage

growth_protocol: Day 15 BU NGST cells were sorted for NKX2-1GFP (as described above) and live positive and negative sorted cells were acquired for scRNA-seq in the Harvard Medical School (HMS) scRNA-seq core laboratory. NKX2-1GFP positive cells were replated in 3D matigel and grown for a further 16 days to day 35. At the selected time points (days 15, 17, 21, 25, 29 and 31) cells derived from directed differentiation of the BU3 NGST iPSC line were stained with calcein blue viability dye and sorted to obtain live cells for scRNA-seq as detailed in the text

extract_protocol:
Cells were captured in the Harvard Medical School (HMS) Single Cell Core for scRNA-seq using inDrops technology, as follows. First cells were assessed for cell number and viability and resuspended in 1000 µL of 15% OptiPrep™ to allow for a homogenous resuspension and reduced clumping. Cell capture and library preparation were performed using a modified version of inDrops protocols (Klein et al., 2015; Plasschaert et al., 2018) involving encapsulation of cells into 3-nl droplets with hydrogel beads carrying barcoding reverse transcription primers.
Following the within-droplet reverse transcription step, emulsions were split into batches of approximately 2,000 cells, frozen at -80C, and subsequently processed as individual RNA-seq libraries (see Table S10). Approximately 4,000 cells for each time point were encapsulated for scRNA-seq

tag: sorting

value: B+

tag: culture media

value: 3D conditions, CKDCI media


title: 6057841123_R06C01


title: lib14_neg_d31

value: B-


title: Liver_adult_3

description: Whole cell RNA from tissue


title: 6057841123_R01C02


title: lib11_pos_d29


title: lib12_pos_d31


title: Retina: Sample 24

city: Iowa City

first: Matthew

last: Harper

state: IA

address: Bldg 42 VAMC

institute: University of Iowa

data_processing:
Enriched RNA pool was fragmented, converted to cDNA and ligated to sequencing adaptors containing indexes using the Illumina TruSeq stranded mRNA sample prep kit (Cat. #RS-122-2101)
Libraries were sequenced on the Illumina HiSeq 4000 genome sequencer using 150 bp paried-end SBS chemistry, then mapped to mm10 whole genome using STAR (v2.5.3a)
We used featureCounts from the subreads package (v1.5.2) to quantify transcripts at the gene level.
Downstream analyses used the R (v3.3.2) packages DESeq2 for differential expression.
Genome_build: mm10

source_name: Retina

extract_protocol:
RNA extraction using Qiagen RNEasy Mini Kit per manufacturers instruction. Illumina TruSeq stranded mRNA Sample Prep Kit (Cat#RS-122-2101) was used with 500 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Each mouse was subjected to a specific blast condition mentioned in SAMPLES section

value: retina

tag: injury

value: sham (5 PSI then 5 PSI)


title: Retina: Sample 23


title: 6057841123_R05C01


title: lib10_pos_d29


title: Retina: Sample 22


title: Liver_E16.5_3

description: Whole cell RNA from tissue


title: 6057841123_R03C01


title: Retina: Sample 19


title: Retina: Sample 20


title: 6057841123_R04C01


title: Preglobular sRNA-seq (biorep 1)

description: Single-end 50 base read sRNA-seq data from first biological replicate of preglobular embryos

value: wild type


title: Kidney_E16.5_3

description: Whole cell RNA from tissue

source_name: Kidney

value: Kidney


title: lib9_pos_d25

value: B


title: Kidney_E16.5_2

description: Whole cell RNA from tissue


title: Retina: Sample 18


title: 6057841123_R03C02


title: lib8_pos_d25


title: lib7_pos_d21


title: lib6_pos_d21


title: Retina: Sample 17


title: Liver_adult_1

description: Whole cell RNA from tissue


title: Retina: Sample 16

value: blast (0 PSI then 20 PSI)


title: lib5_pos_d17


title: 6057841105_R05C02


title: Kidney_adult_3

description: Whole cell RNA from tissue


title: Kidney_adult_2

description: Whole cell RNA from tissue


title: 6057841105_R04C02


title: Retina: Sample 15


title: Kidney_adult_1

description: Whole cell RNA from tissue


title: 6057841105_R03C02


title: lib4_pos_d17


title: Heart_E16.5_3

description: Whole cell RNA from tissue

source_name: Heart

value: Heart


title: lib3_neg_d15

value: Ve-


title: 6057841123_R01C01

source_name: buccal swab genomic DNA


title: Retina: Sample 14


title: Kidney_E16.5_1

description: Whole cell RNA from tissue


title: 6057841105_R02C02

value: F


title: 6057841105_R06C02


title: lib2_pos_d15

value: Ve+


title: Retina: Sample 13


title: Heart_E16.5_2

description: Whole cell RNA from tissue


title: 6057841105_R01C02


title: lib1_pos_d15


title: Retina: Sample 12


title: Heart_E16.5_1

description: Whole cell RNA from tissue


title: 6057841105_R06C01


title: Retina: Sample 11


title: Retina: Sample 10


title: Heart_adult_3

description: Whole cell RNA from tissue


title: 6057841105_R05C01


title: Retina: Sample 9


title: Heart_adult_2

description: Whole cell RNA from tissue


title: 6057841105_R04C01


title: infected - library 2

data_processing:
Data was demultiplexed with inDrops pipeline
Mapping done with Bowtie
Differentiall expression and downstream analysis on Seurat
Lentiviral barcode analysis done with LARRY pipeline
Genome_build: GRCh38
Supplementary_files_format_and_content: The processed data contains the counts matrices for the mRNA expression libraries and the lentiviral barcodes detected on the corresponding cells (files starting with "T" from tag)

source_name: differentiated iPSC

growth_protocol: BU3 NGST human iPSCs were differentiated using previously described protocols until infection on D17 (Jacobs et al., 2017). After infection on D17, cells were resuspended in 3D Matrigel and fed every other day with CK+DCI supplemented with Rock Inhibitor.

extract_protocol:
Cell capture and library preparation were performed as described using a modified version of inDrops protocols(Klein et al., 2015; Plasschaert et al., 2018). Prior to library preparation, RNA fractions generated from each population were split in half, with one half being used for standard library prep and the other half for targeted lineage barcode enrichment.
To enrich for lineage barcodes, library preparation was modified as previously described (Weinreb et al., 2019) by skipping RNA fragmentation, priming the RT reaction using a barcode-specific primer (TGAGCAAAGACCCCAACGAG), introducing an extra PCR step using a targeted primer (8 cycles using Phusion 2X master mix; Thermofisher; primer sequence = TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG NNN Ntaa ccg ttg cta gga gag acc atat), and 1.2X bead purification (Agencourt AMPure XP). All targeted and non-targeted final libraries were pooled at equimolar ratios and sequenced using Illumina NextSeq 500 Sequencing (75 Cycles, 75bp Single Read sequencing).

treatment_protocol: On D17 3D Matrigel was broken down using Dispase and cells were resuspended in CK+DCI with polybrene and either no virus (MOI 0) or the lentiviral barcode at a multiplicity of infection of 10 (MOI 10). Cell suspension was placed at 37C and incubated for 4 hours with occasional flicking. At the end of this period cells were replated as small cell clumps in 3D Matrigel.

value: day 27

value: CKDCI

tag: isolation method

value: FACS sorted + indrops


title: Heart_adult_1

description: Whole cell RNA from tissue

tag: tissue

value: adult


title: Retina: Sample 8

value: preconditioned (5 PSI then 20 PSI)


title: control - not infected


title: 6057841105_R03C01


title: Brain_E9.5_3

description: Whole cell RNA from tissue

source_name: Whole head

value: Whole head


title: infected - library 3 (enriched for GFP+ cells)


title: 6057841105_R02C01

value: M


title: Retina: Sample 7

value: retina


title: Brain_E9.5_2

description: Whole cell RNA from tissue


title: 6057841105_R01C01


title: Brain_E9.5_1

description: Whole cell RNA from tissue


title: Retina: Sample 6


title: Retina: Sample 5


title: iAEC2 alveolospheres

description: iAEC2, SFTPC Tomato positive

data_processing:
The sample was processed using the standard Cellranger v.3 pipeline
Reads were mapped to GRCh38
Downstream analysis and QC done on Seurat
Differential expression using MAST models
Genome_build: GRCh38
Supplementary_files_format_and_content: the processed files are the standard output from the cellranger count pipeline: expression sparse matrix, gene list and barcode list

source_name: iPSC derived

growth_protocol: To test for long term maintenance of the lung epithelial program in iPSC-derived alveolospheres we used our published iAEC2 differentiation protocol (Jacob et al., 2017) with extensive details including serial alveolosphere passaging techniques detailed in Jacob et al. 2019, Nature Protocols, in press.

extract_protocol:
single cells were captured for sequencing library prepration using a 10X Chromium
10X chromium chemistry v.2

treatment_protocol: SPC2 iPSCs were differentiated until day 16 when primordial lung progenitors were sorted based on CD47hi/CD26lo gating (Hawkins et al., 2017). After replating these purified progenitors in 3D Matrigel cultures in “CK+DCI” media, the resulting epithelial spheres were passaged without further sorting on day 37 and CHIR was briefly withdrawn from days 40-44 to achieve iAEC2 maturation (Jacob et al., 2017). Then CHIR was added back for the duration of the experiment. On day 51 SFTPCtdTomato+ cells were sorted and replated as alveolospheres with subsequent passaging without further cell sorting performed on days 65, 82, 101, and 114

value: day 115

value: 3D conditions in CK+DCI media

value: Chromium 10X


title: Brain_E16.5_3

description: Whole cell RNA from tissue

source_name: Brain

value: Brain


title: UPF1 ChIP-seq

city: Incheon

first: Donghee

last: Kang

address: 100 Inha-ro, Michuhol-gu

department: Medicine

country: South Korea

institute: Inha University

library_source: genomic

data_processing:
Reads mapping on the reference genome (human hg19)  using Bowtie2 version 2.3.1
Cross correlation analysis using SPP version 2.0
Peaks were called using MACS
Genome_build: hg19
Supplementary_files_format_and_content: fastq files include raw reads of sequencing and .xls file is a processed data file that UPF1 peaks

source_name: Breast cancer cells

growth_protocol: MCF7 cells were cultulred in Dulbecco's modified Eagle's medium (DMEM) (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (Tissue Culture Biologies, Long Beach, CA, USA) and 1% penicillin and streptomycin solution (WelGENE) at 37°C in a 5% CO2 incubator.

extract_protocol:
The DNAs and RNAs pulled down with proteins were purified by phenol-chloroform extraction and precipitated in ethanol.
ChIP-seq libraries were constructed using TruSeq Strandard library protocols.

treatment_protocol: For ChIP, cells were lysed in NET-2 buffer containing 100 U of RNase Inhibitor  (Invitrogen, Carlsbad, CA, USA) and preclearing with Protein A-Sepharose beads (GE Healthcare Bio-Science AB, Uppsala, Sweden). After preclearing, the lysates were immunoprecipitated with specific antibodies or IgG along with beads.

tag: cell type

value: epithelial

value: mammary gland, breast; derived from metastatic site:pleural effusion

tag: disease

value: adenocarcinoma

value: 69 years adult

tag: chip antibody

value: Anti-RENT1 (UPF1) (Bethyl Laboratories, A301-902A)


title: infected - library 1

value: day 27


title: Linc-ASEN ChIRP-seq

data_processing:
Library strategy: ChIRP-seq
Reads mapping on the reference genome (human hg19)  using Bowtie2 version 2.3.1
Cross correlation analysis using SPP version 2.0
Peaks were called using MACS
Genome_build: hg19
Supplementary_files_format_and_content: fastq files include raw reads of sequencing and .xls file is a processed data file that Linc-ASEN peaks

extract_protocol:
Beads were resuspended in 100 μl of 2x Laemmli sample buffer and boiled for 5 min, after which they were subjected to trizol:chloroform extraction.
ChIRP-seq libraries were constructed using TruSeq Strandard library protocols.

treatment_protocol: ChIRP assays were performed using method described in Chu et al., 2011. Briefly, three 3' biotinylated probes targeting the full-length of the Linc-ASEN sequence were designed. MCF7 cells were then sonicated in NET-2 buffer and pre-cleared with streptavidin beads (Sigma Aldrich) at 4°C for 1 hr with end-over-end rotation.  After pre-clearing, 2 μl per 100 pmol of each probe were added and incubated at 4°C for 2 hrs. Beads:biotin-probes:RNA:chromatin were captured with streptavidin beads (Sigma Aldrich).

tag: chirp

value: Linc-ASEN biotin-labelled probes


title: 6057841102_R05C02

value: F


title: Retina: Sample 4


title: 6057841102_R04C02


title: MCF7-Linc-ASEN siRNA

data_processing:
Basecalls performed using CASAVA version 1.8.2
Sequencing quality check and count perfomed using FastQC version 0.11.3
Sequenced reads were trimmed using CutAdapt version 1.8
Alignment of reads to the reference genome (human hg19) was performed using STAR version 2.5.0
Expression profiling and differentially expressed gene (DEG) analysis using Cufflinks version 2.2.1.
Genome_build: hg19
Supplementary_files_format_and_content: fastq files include raw reads of sequencing and .xlsx file is a processed data file that contains DEG

extract_protocol:
Total RNAs were extracted from MCF7 cells using TRIzol Reagent (Molecular Research Center, Cincinnati, OH, USA)
mRNA libraries were prepared for sequencing using Truseq Standard mRNA kit (Illumina)

treatment_protocol: Transfection of control siRNA or Linc-ASEN siRNA in MCF7 cells.

tag: transfection

value: Linc-ASEN siRNA


title: Brain_E16.5_2

description: Whole cell RNA from tissue


title: Retina: Sample 3

value: preconditioned (5 PSI then 20 PSI)


title: Retina: Sample 2


title: MCF7-Control siRNA

value: control siRNA


title: ZRF1 KD replicate 3

data_processing:
Reads were demultiplexed and fastq files were generated with Illumina bcl2fastq-1.8.4
Fastq files were mapped with STAR_2.5.2b onto the Ensembl GRCh38 version 84
Secondary alignments were filtered with samtools 1.3
Counts for every gene were calculated with featureCounts from the Subread package version 1.5.1, using the gene model from Ensembl GRCh38 version 84
Counts were normalized and modeled with DESeq2_1.14.1
Genome_build: GRCh38
Supplementary_files_format_and_content: tab separated file containing counts per gene

source_name: MCF7 breast cancer cell line

growth_protocol: DMEM + 10% FBS + L-Glutamine + P/S

extract_protocol:
For each replicate, RNA was isolated from 7 days old organoids derived from control and shZRF1 MCF7 cells using Direct-zol RNA MiniPrep kit (Zymo Research).
NGS library preparation was performed with Illumina's TruSeq stranded RNA LT Sample Prep Kit following Illumina’s standard protocol. Libraries were prepared with a starting amount of 900 ng and amplified in 10 PCR cycles.

treatment_protocol: MCF7 cells are maintained in normal growth medium in 1% agarose coated 96 well plates during 7 days. Every 2 days, medium was replenished.

tag: cell line

tag: shRNA


title: Brain_adult_3

description: Whole cell RNA from tissue

value: adult


title: 6057841102_R03C02


title: Brain_E16.5_1

description: Whole cell RNA from tissue


title: Retina: Sample 1

tag: strain

value: retina


title: 6057841102_R02C02

tag: age


title: ZRF1 KD replicate 2


title: 6057841102_R06C02

tag: gender

value: M


title: Brain_adult_2

description: Whole cell RNA from tissue


title: SI rep3

city: Beijing

first: Ziqiang

last: Wang

address: North Street of Zhongguancun, Haidian Distric

institute: Tsinghua University

data_processing:
Filtering of Clean Reads:1) removing reads containing adapters; 2) removing reads containing more than 10% of unknown nucleotides (N); 3) removing low quality reads containing more than 50% of low quality (Q-value≤ 2 0 bases)
Short reads alignment tool Bowtie 2 was used for mapping reads to ribosome RNA (rRNA) database
The rRNA removed reads of each sample were then mapped to reference genome by TopHat 2 2 (version 2.0.3.12)
The reconstruction of transcript s was carried out with software Cufflinks and TopHat 2
Gene abundances were quantified by software RSEM
Genome_build: hg19
Supplementary_files_format_and_content: xls file containing raw read counts and data with values for all genes

source_name: Epidermis

growth_protocol: HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (GIBCO) supplemented with 10% fetal bovine serum in 5% CO2 humidity incubator at 37°C

extract_protocol:
Standard TriZol reagent was used to isolate total RNA from each sample
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: HeLa cells were transfected with B2M siRNAs or negative control siRNAs for 48 hours. Then, the cells were infected with HSV-1 at an MOI of 1 for 4 hours and were harvested for total RNA isolation.

value: cervix

tag: treatment

value: B2M knockdown with siRNAs


title: ZRF1 KD replicate 1


title: SI rep2


title: Brain_adult_1

description: Whole cell RNA from tissue

value: Brain

tag: developmental stage


title: 6057841102_R06C01


title: 6057841102_R01C02


title: CHIP_INPUT_TKO_14d_2i

city: Edinburgh

first: John

middle: Paterson

last: Thomson

address: Crewe Road

department: MRC Human Genetics Unit

country: United Kingdom

institute: University of Edinburgh

description: mapped reads (mm10) were binned across 200bp windows.

data_processing:
Reads mapped to mm10 mouse build in the cloud using TMAP software
Data binned into 200bp windows across the genome
Data converted into mm9 build and into a wiggle (.wig) file
data normalised by read count
enriched (5mC) datasets normalised to matched input dataset to generate enrichment over background dataset
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph file

source_name: DNMT 1/3a/3b null mutant mouse J1 ESCs converted in 2i

growth_protocol: J1 mESCs were cultured in Dulbecco's Modified Eagle's medium (DMEM) high-glucose (Life Technologies™) supplemented with 15% FCS (Sigma), 1x Non-essential amino acids (Sigma), 1mM Sodium Pyruvate (Sigma), 2mM L-glutamine (Life Technologies™), 50µg/ml Penicillin/Streptomycin, 100μM beta-mercaptoethanol (Gibco) and 500U/ml ESGRO LIF ESG1107 (Millipore). J1 mESCs were grown on Mitomycin C inactivated SNLP feeder cells, 2i cells were grown feeder free on 0.2% gelatin-coated plates or flasks following culturing conditions as described in Ying et al 2008.

extract_protocol:
Sheared gDNA was enriched using antibodies of choice. prior to enrichment through magnetic IgG beads . Following a series of buffer washes the DNA is then released through proteinase K digestion of the antibody and the DNA is subsequently cleaned up using Qiagen QIAquick PCR purification kits (Qiagen) and eluting in 20ul purified water. Enriched DNA was then subjectedto 18 cycles of whole genome amplification using using an enhanced amplification kit optimised for next generational sequencing (Sigma-Aldrich SeqPlex DNA Amplification Kit).
Individual libraries were generated from 100ng each of 5mc enriched and input DNA for each sample, using the Ion XpressPlus Fragment Library Kit (life Technologies™). The DNA was end repaired, purified and then ligated to Ion-compatible barcoded adapters (Ion Xpress™ Barcode Adapters 1–96: Life Technologies™), followed by nick-repair to complete the linkage between adapters and DNA inserts. The adapter-ligated library was then amplified (10 cycles) and finally size-selected using two rounds of AMPure XP bead capture to size‐select fragments approximately 100–250 bp in length. An equimolar pool of barcoded libraries was prepared at 100pM; each pool contained an meDIP enriched and corresponding input sample. 8pM of the pooled library was added into an emulsion PCR based template reaction; in this reaction the fragments generated during the library prep were attached to Ion sphere particles (ISPs) and clonally amplified. This process was carried out using the Ion One Touch 2 system and the Ion P1 Template OT2 200 Kit.

tag: culture condition

value: 14 day 2i media

tag: strain/cell type

value: J1 - Dnmt1/3a/3b triple knock out

value: mESC

tag: antibody

value: none (input)


title: SI rep1


title: Control replicate 3, sh empty vector

value: Control


title: CHIP_INPUT_TKO_serum

source_name: DNMT 1/3a/3b null mutant mouse J1 ESCs

value: Serum


title: 6057841102_R04C01


title: Control replicate 1, sh empty vector


title: NC rep3

value: negative control


title: NC rep2


title: 6057841102_R02C01


title: CHIP_INPUT_3B3_serum

source_name: DNMT 3B3/3L mutant mouse J1 ESCs

value: J1 - Dnmt3b & 3l hyperactive


title: NC rep1


title: 6057841135_R05C01

value: F


title: 6057841102_R01C01


title: 6057841100_R06C02


title: DIP_INPUT_3B3_serum


title: Subject 12005 Pre

city: Orlando

first: Rick

last: Vega

state: FL

address: 303 E. Princeton

institute: AdventHealth

data_processing:
Reads were mapped using tophat 2.0.1
mRNA quantification and normalization was performed with Partek Genomic Suite 6.6
Differential expression was calculated using Bioconductor DEseq R package as described in Anders S, Huber W (2010). “Differential expression analysis for sequence count data.” Genome Biology, 11, R106.
Genome_build: hg38

source_name: Skeletal muscle biopsy

extract_protocol:
Muscle biopsies were obtained using a Berstrom needle. The biopsy was trimmed of visible adipose tissue and flash frozen in liquid nitrogen. Total RNA was isolated using the RNeasy fibrous tissue kit.
1microgram of total RNA was utilized for library construction using the Illumina Truseq stranded Total RNA library preparation kit.

tag: group

value: before bed rest

value: Vastus lateralis


title: DIP_INPUT_3B3_14d_2i

source_name: DNMT 3B3/3L mutant mouse J1 ESCs converted in 2i


title: 6057841102_R05C01

last: Kobor

address: 950 West 28th Avenue

institute: Centre for Molecular Medicine and Therapeutics / University of British Columbia


title: Subject 12003 Post

value: 10 days after bed rest

tag: tissue


title: Subject 12005 Post


title: 3% DSS 4

type: RNA

description: mirRNA levels in colonic crypts from mice treated with 3%DSS drinking water for 7 days

hyb_protocol: Cy3-pCp labelled RNA was dryed and then hybridized using Agilent Microarray Hybridization Chamber Kit (G2534A) according to the  manufacturer's instructions.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x60k array slides (AgilentG3_miRNA).

data_processing: The scanned images were analyzed with Agilent Feature Extraction(AFE) software version 10.7.1.1 using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: colonic crypts

label_protocol: Diluted total RNA was dephosphorylated using MicroRNA Spike-In Kit according to the manufacturer's instructions, followed by denaturation, ligation using T4 RNA ligase to label Cyanine-3 (Cy3)-pCp.

extract_protocol: Following euthanasia of DSS-treated mice, the entire colon was removed, everted, and rinsed 5 times with cold PBS (supplemented with 2% (vol/vol) penicillin and streptomycin). The rinsed colon was immersed into chilled (0-4 ºC) cell recovery solution buffer (354253, Corning, NY, USA) for 30-40 min; the resulting cell suspension was filtered through 70-μm strainers and rinsed with basic cell culture medium (DMEM/F12 (GibcoTM, Waltham, MA) supplemented with 1% (vol/vol) penicillin and streptomycin (GibcoTM), 1% glutamax (Gibco GibcoTM), and 1% HEPES (GibcoTM)) until the supernatant remained clear after centrifugation at 200xg for 2-3 min. RNA was harvested using Trizol reagent. Total RNA  were extracted by miRNeasy Mini Kit and then used in The Agilent miRNA Microarray system.

treatment_protocol: C57BL/6 mice (male,8-week-old ) were provided with free access to normal drinking water or 3% DSS (160110, MW=36-50 kD; MP Biomedicals, Santa Ana, CA, USA) in the drinking water for 7 days; the first day of DSS access was designated Day 0.

value: male

tag: cell type

value: colonic crypt cells

value: 3% DSS for 7 days


title: 3% DSS 3


title: 3% DSS 5


title: 6057841100_R05C02

value: M


title: Control replicate 2, sh empty vector


title: CHIP_INPUT_3B3_14d_2i


title: 6057841102_R03C01


title: H3K27me3_ChIPseq_TKO_serum

value: Anti H3K27me3 - Millipore (cat # 07-449)


title: H3K27me3_ChIPseq_TKO_14d_2i

value: 14 day 2i media


title: Subject 12003 Pre


title: H3K27me3_ChIPseq_3B3_14d_2i


title: 6057841100_R04C02


title: Subject 11006 Post


title: 6057841100_R03C02


title: H2O 5

description: mirRNA levels in colonic crypts from mice treated with normal water


title: H3K27me3_ChIPseq_3B3_serum


title: 6057841100_R02C02


title: in vitro biofilm, 33-6_RNA_S8

city: Columbus

first: Alistair

last: Harrison

state: OH

address: 700 Children's Drive

department: Center for Microbial Pathogenesis

institute: The Research Institute at Nationwide Children's Hospital

description: nontypeable Haemophilus influenzae

data_processing:
Image analysis and base calling were performed with XXSequence Analysis Viewer tool
Each sample was aligned to the 86-028NP  reference genome (NCBI; GCA_000012185.1) using the BWA-MEM aligner
Feature coverage counts were calculated using HTSeq and differentially expressed features were calculated using DESeq2
Genome_build: NCBI; GCA_000012185.1
Supplementary_files_format_and_content: Raw and normalized abundance measurements

organism: Haemophilus influenzae

source_name: in vitro biofilm

growth_protocol: Suspensions of bacteria in iron chelated defined iron source (DIS) medium were adjusted to an OD490 of ~0.65 , diluted 10-fold and subcultured into nitric acid-washed 15 ml round bottom glass tubes containing pre- warmed DIS medium with 2 mg/ml of  heme. After static growth for 24 hours at 37˚C in 5% CO2 . Cultures were then equilibrated to an OD490 of ~0.37 and diluted to a concentration of ~ 1E+07 CFU/ml in DIS containing 2 mg /ml of heme . Fifty microliters were then added to each well of a Nunc Lab-Tek 8- well chamber slide containing 200 µl of DIS supplemented with 2mg/ml of  heme. The chamber slide was then incubated at 37˚C with 5% CO2 for 48  with a medium change at 24 hours.

extract_protocol:
Biofilms were scrapped form the chamber suraces and added directly to hot phenol. RNA was purified as described in Santana et al (2014; PMID:25157846)
RNA libraries were prepared for sequencing using standard Illumina protocols

value: 86-028NPiccD21V (persister strain)

tag: sample type

value: in vitro biofilm


title: Subject 11006 Pre


title: H2O 4


title: in vitro biofilm, 33-5_RNA_S7

description: nontypeable Haemophilus influenzae


title: 6057841100_R01C02

city: Vancouver

first: Michael


title: hMeDIP_seq_3B3_14d_2i

value: mESC

value: Anti 5-hmC - Active Motif (cat# 39769)


title: H2O 3


title: in vitro biofilm, 33-2_RNA_S6

description: nontypeable Haemophilus influenzae


title: hMeDIP_seq_3B3_serum

value: Serum


title: 6057841100_R06C01


title: 6057841100_R05C01


title: in vitro biofilm, 33-1_RNA_S5

description: nontypeable Haemophilus influenzae


title: hMeDIP_seq_J1_14d_2i

source_name: mouse J1 ESCs converted in 2i