title: Ecoli_ΔgreA_ΔgreB_NETseq

description: library strategy: NET-seq

value: ΔgreA ΔgreB


title: Ecoli_ΔgreA_NETseq

status: Public on May 05 2014

type: SRA

city: San Francisco

first: Matthew

middle: H

last: Larson

state: CA

address: 499 Illinois St

country: USA

institute: Grail, Inc.

description: library strategy: NET-seq

library_source: transcriptomic

data_processing:
Basecalls performed using Casava versions 1.6 or 1.7.
Sequenced reads were trimmed for adaptor sequence.
Trimmed reads were aligned using Bowtie v0.12.7 against the reference genome using parameters -a -v 3 -m 1
Bowtie alignments against the reference genome were converted to wiggle files. The position of each alignment was mapped to the 3' end of the nascent transcript.
Genome_build: E. coli: NC_000913
Supplementary_files_format_and_content: wiggle files with two columns: first column containing chromosome positions and second column containing the number of reads mapped to the position (see publication for details).

molecule: total RNA

organism: Escherichia coli

source_name: Bacteria

growth_protocol: For each sample, 500 mL liquid cultures were grown in 2.8 L flasks with shaking (180 rpm) at 37 °C from an OD (600 nm) 0.05 to OD 0.45 ± 0.05. Cells were grown in MOPS EZ rich defined medium with 0.2% glucose (Teknova).

extract_protocol:
Cell culture was rapidly filtered in 250 mL increments at 37 °C over 0.22 μm nitrocellulose ﬁlters (GE) and frozen in liquid nitrogen to simultaneously halt all transcriptional progress. Frozen cells (100 μg) were pulverized on a Qiagen TissueLyser II mixer mill 6 times at 15 Hz for 3 min in the presence of 500 μL frozen lysis buffer (20 mM Tris pH 8, 0.4% Triton X-100, 0.1% NP-40, 100 mM NH4Cl, 50 U/mL SUPERase•In (Ambion)) and 1X protease inhibitor cocktail (Complete, EDTA-free, Roche), supplemented with 10 mM MnCl2. The lysate was resuspended on ice by pipetting. RQ1 DNase I (110 U total, Promega) was added and incubated for 20 min on ice. The reaction was quenched with EDTA (25 mM final), which releases polysomes from the transcript and reduces contamination from ribosomal RNA and ribosome-associated tRNAs. The lysate was clarified at 4 °C by centrifugation at 20,000 g for 10 min. The lysate was loaded onto a PD MiniTrap G-25 column (GE Healthcare) and eluted with lysis buffer supplemented with 1 mM EDTA. For nascent RNA purification, the clarified lysate was added to 0.5 mL anti-FLAG M2 affinity gel (Sigma Aldrich). The affinity gel was washed twice with lysis buffer supplemented with 1 mM EDTA before incubation with the clarified lysate at 4 °C for 2.5 h with nutation. The immunoprecipitation was washed 4 × 10 mL with lysis buffer supplemented with 300 mM KCl, and bound RNAP was eluted twice with lysis buffer supplemented with 1 mM EDTA and 2 mg/mL 3XFLAG peptide (Sigma Aldrich). Nascent RNA was purified from the eluate using the miRNeasy kit (Qiagen).
The full library construction protocol was described in detail previously (Churchman et al., Nature 2011). Purified nascent RNA was ligated to a 5' adenylated DNA oligo and fragmented to generate RNA ranging from 30-100 nt. The RNA was reverse transcribed, and the single-stranded DNA circularized and PCR amplified.

tag: strain background

tag: genotype/variation

value: ΔgreA

tag: molecule subtype

value: Nascent RNA

tag: media

value: MOPS EZ Rich Defined Media


title: Ecoli_ΔgreB_NETseq

description: library strategy: NET-seq

value: ΔgreB


title: Ecoli_WT_NETseq

description: library strategy: NET-seq

data_processing:
Basecalls performed using Casava versions 1.6 or 1.7.
Sequenced reads were trimmed for adaptor sequence.
Trimmed reads were aligned using Bowtie v0.12.7 against the reference genome using parameters -a -v 3 -m 1
Bowtie alignments against the reference genome were converted to wiggle files. The position of each alignment was mapped to the 3' end of the nascent transcript.
After alignment and mapping of the reads, pause detection was performed using custom scripts written in Python 2.7. Positions with counts greater than four standard deviations above the mean were scored as pauses.
Genome_build: E. coli: NC_000913
Supplementary_files_format_and_content: wiggle files with two columns: first column containing chromosome positions and second column containing the number of reads mapped to the position (see publication for details).
Supplementary_files_format_and_content: The fas.txt contains two lines for each pause; the first line denotes the gene, strand, pause index, and relative peak height. The second line denotes the sequence 15 nt upstream and downstream of the pause position.

value: wild type


title: D14_mRNA-seq_replicate_2

city: Ann Arbor

first: Lihong

last: Shi

address: 3608 BSRB, 109 Zina Pitcher Place

institute: University of Michigan

data_processing:
Identification and quantification of previously annotated transcripts: For mRNA-seq data analysis, we aligned the raw reads from ELAND to the human genome (build hg18) using TopHat (version 1.3.3) at the default settings. We then used Cufflinks (version 1.2.1) to identify previously annotated transcripts, and then to determine gene- and isoform-specific expression levels of those transcripts in units of fragments per kilobase of exon per million fragments mapped (FPKM). The results from two biological replicates were merged using Cuffmerge, and then Cuffdiff was used to identify differentially expressed transcripts at the different stages (day 4, 8, 11, or 14).
Identification of novel transcript isoforms and intergenic or intronic transcripts: To identify novel transcript isoforms as well as intergenic or intronic transcripts from the mRNA- seq data sets, we performed de novo assembly of the transcripts (without supplying gene model annotations) by using Cufflinks to processes output BAM files from TopHat. The results from replicates were then merged using Cuffmerge, and then Cuffdiff was used to perform differential expression analysis as described above.
Genome_build: hg18
Supplementary_files_format_and_content: transcript abundance at each differentiation stage

molecule: polyA RNA

organism: Homo sapiens

source_name: in vitro differentiated erythroid cells

growth_protocol: CD34+ cells, which were mobilized in and isolated from the peripheral blood of healthy donors, were purchased from the Fred Hutchinson Cancer Research Center. The cells were grown and differentiated ex vivo into the erythroid lineage by a two-phase culture method. Briefly, during the first phase (from days 0 to 8), the cells were cultured in IMDM basic medium (Biochrom) supplemented with 40 µg of inositol/ml, 10 µg of folic acid/ml, 160 µM monothioglycerol, 120 µg of iron-saturated human transferrin/ ml, 10 µg of insulin/ml, 900 ng of ferrous sulfate/ml, 90 ng of ferric nitrate/ml, 1 µM hydrocortisone, and 5 ng of interleukin-3 (IL-3)/ml, which were all pur- chased from Sigma-Aldrich, and 200 mM L-glutamine (Invitrogen), 1% deionized bovine serum albumin (Stemcell Technologies), 100 ng of stem cell factor (Prospec)/ml, and 3 IU of erythropoietin/ml, which was purchased from Amgen. During the second phase (day 8 through day 14), the cells were cultured in the same medium without hydrocortisone or IL-3.

extract_protocol:
Total RNA was purified with an ISOGEN (Nippon Gene) reagent from primary human erythroid cells after 4, 8, 11 or 14 days of ex vivo differentiation of CD34+ cells. The integrity and quality of RNA samples were assessed by the RNA integrity number (RIN) determined with an Agilent 2100 Bioanalyzer (Agilent Technologies). RINs of all the RNA samples used in this study were 10, indicating RNA of the highest integrity.
The construction of the mRNA-seq libraries was performed according to standard instructions from Illumina. In brief, poly-A+ RNA was purified by poly-dT oligo-attached magnetic beads from 10 µg total RNA extracted from primary human erythroid cells after 4, 8, 11 or 14 days of ex vivo differentiation, and then sheared into short fragments of 200-300 bp in length by heating in the presence of divalent Zn2+ cations. These RNA fragments were reverse-transcribed using random primers into double-stranded cDNA fragments, which were end-repaired and then subjected to addition of a single adenine followed by ligation with an Illumina adapter to both ends. After PCR amplification, cDNA fragments approximately 200 bp in length were selected to generate a library to be sequenced in a paired-end 72-bp sequencing format using the Illumina Genome Analyzer IIx system in the DNA sequencing core facility of the University of Michigan. Two replicate RNA samples at each differentiation stage (day 4, 8, 11, and 14) were prepared from independent CD34+-cell cultures to generate two biological replicates of mRNA-seq libraries and data sets.

tag: cell type

value: in vitro differentiated erythroid cells

tag: cell composition

value: orthochromatic erythrolast and 30% reticulocyte

tag: differentiation day


title: D11_mRNA-seq_replicate_2

value: including basophilic and polychromatic erythroblast cells


title: D8_mRNA-seq_replicate_2

value: majority in proerythroblast stage


title: D4_mRNA-seq_replicate_2

value: immature erythroid cells


title: 129S1_MA15_D4_5

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

data_processing:
Reads were basecalled using SCS version 2.9 software and processed using version CASAVA 1.8.2
Reads were mapped against specific CC founder strain transcriptome with SOAPaligner/soap2 and following command : soap -m 20 -x 500 -r 2 -v 2
To determine fragment count on gene level from SOAP output, we used a custom script in Java reproducing HTSeq paired and strand-specific union mode
Technical replicates were summed
Three samples were excluded based on their low raw gene counts distribution ("PWK_MA15_D2_116", "NOD_MA15_D2_17", "CAST_MA15_D2_99").
Data normalization was performed by scaling as implemented in the DESeq bioconductor package
Genome_build: CC founder pseudogenomes
Supplementary_files_format_and_content: tab-delimited text files include scaled/normalized gene counts for each sample

organism: Mus musculus

source_name: lung

growth_protocol: Whole Animal

extract_protocol:
Qiagen miRNeasy Isolation Kit from Trizol homogenates
Illumina TruSeq Total RNA

treatment_protocol: Intranasally infected or mock infected

tag: strain

tag: virus

tag: day-post-infection


title: D4_mRNA-seq_replicate_1


title: D8_mRNA-seq_replicate_1


title: 129S1_MA15_D2_1


title: D11_mRNA-seq_replicate_1


title: STT5831_Breast_024_normal

status: Public on May 04 2014

city: Stanford

first: Robert

middle: B.

last: West

address: 300 Pasteur Drive

department: Pathology

institute: Stanford School of Medicine

data_processing:
3SEQ libraries were sequenced with Illumina GAIIx machines to obtain 36-base directional, single-end sequence reads.  The first 25 bases of the sequence reads were mapped to hg18 with a two-mismatch allowance using ELAND (Illumina).
Sequence reads were further filtered to remove mapping artifacts caused by ambiguous mapping by requiring a posterior probability of at least 0.8 for the best alignment, where posterior probability was calculated as the ratio of the likelihood of the best alignment to the sum of the likelihoods of all alignments.  Likelihood was taken from a binomial model of sequencing errors with constant substitution rate 0.01.
Reads within exons of RefSeq genes (n=22,775 genes; downloaded July 2011 from the University of California Santa Cruz Genome browser) were tallied to obtain the total number of reads per gene for each sample (Raw_counts_Refseq_genes.txt).
Genes with an average of less than 1 read per sample (< 72 reads across all samples) were removed from the dataset, resulting in a dataset of 15,748 genes.  Read counts were transformed using Anscombe transformation to stabilize variance, and then normalized using sequencing depths estimated by PoissonSeq using package SAM 2.0.  The resulting normalized data are roughly Gaussian-distributed and the values are comparable across samples, resembling microarray data (Normalized_RefSeq_genes.txt).
3SEQ peaks previously identified for 1,065 known lncRNAs and 1,071 novel transcripts were profiled within our samples by counting sequencing reads falling within the previously defined peak coordinates (Brunner et al, Genome Biol 2012, 13:R75) (Raw_counts_lncRNAs.txt).
Peaks with an average of less than 1 read per sample (< 72 reads across all samples) were removed from the dataset, resulting in a dataset of 1,839 lncRNAs and novel transcripts, which was then transformed and normalized as described above (Normalized_lncRNAs.txt).
Genome_build: hg18
Supplementary_files_format_and_content: Raw_counts_Refseq_genes.txt:  Matrix table of raw sequencing reads for each RefSeq gene for each sample (22,775 genes x 72 samples).
Supplementary_files_format_and_content: Normalized_RefSeq_genes.txt:  Matrix table of normalized sequencing data for each RefSeq gene for each sample (15,748 genes x 72 samples).
Supplementary_files_format_and_content: Raw_counts_lncRNAs.txt:  Matrix table of raw sequencing reads for each lncRNA/novel transcript for each sample (2,136 transcripts x 72 samples).
Supplementary_files_format_and_content: Normalized_lncRNAs.txt:  Matrix table of normalized sequencing data for each lncRNA/novel transcript for each sample (1,839 transcripts x 72 samples).

source_name: normal breast

extract_protocol:
Multiple 2 mm-diameter core punches were taken from FFPE tissue blocks to collect dense areas of neoplasia and adjacent normal breast epithelial content separately.  When ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC) were present, these were also cored.  Three to ten tissue cores were pooled for each diagnosis per patient, and tissue cores were re-embedded in paraffin and re-evaluated histologically for lesion purity by sectioning length-wise along the core.  Only samples which possessed >90% of luminal cells with the appropriate diagnosis within the epithelial compartment were included among our samples.  Adjacent normal cores contained no identifiable pathologies, and cancer cores possessed less than 5% early neoplasia or normal epithelial cells.  In all cases stromal cells were present in the core samples.  DNA and total RNA were purified from paraffin slices of the embedded tissue cores following deparaffination with a xylene incubation, ethanol wash, and a protease digestion, using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion/Life Technologies; catalog #1975).
3SEQ libraries were prepared from as little as 5 ug total RNA.  RNA was enriched for 3’ ends by using the Oligotex mRNA mini Kit (QIAgen).  The 3’- poly(A)-enriched RNA pools were made into directional 3SEQ Illumina sequencing libraries as follows.  RNA that was not sufficiently fragmented was heat-sheared to a size of approximately 100-200 bases by incubation with First Strand Buffer (Invitrogen/Life Technologies) and oligo-dT-P7 primer (5’- CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC TTT TTT TTT TTT TTT TTT TTT TTT TVN -3’) at 85oC for 3-5 minutes, depending on the extent of fragmentation required.  The mixture was cooled to 50oC and first strand cDNA synthesis was performed in a 20 ul reaction using Superscript III Reverse Transcriptase (Invitrogen/Life Technologies) for 1 hour at 50oC.  Second strand cDNA was synthesized using Second Strand Buffer (Invitrogen/Life Technologies), DNA ligase (Invitrogen/Life Technologies), DNA polymerase I (New England Biolabs), and RNaseH (Epicentre Biotechnologies/Illumina), and purified using the QIAquick PCR Purification Kit (QIAgen).  3’ end repair and modification used dATP, Klenow exo (New England Biolabs) and Klenow Buffer, and was purified using the QIAgen MinElute PCR Purification Kit (QIAgen).  cDNA was then ligated to the double-stranded P5 Linker (Adapter Oligo Mix; Illumina) for 15 minutes at 22oC.  SPRI bead purification (Agencourt Biosciences/Beckman Coulter) was performed both prior to and following PCR amplification of the linker-ligated cDNA using 5' - AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T - 3' and 5' - CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC - 3' primers and the Phusion PCR Master Mix (New England Biolabs) using a 15 cycle program (98oC for 30 sec; 15 cycles of 98oC for 10 sec, 65oC for 30 sec, 72oC for 30 sec; 72oC for 5 min).  The libraries were then size selected for 200–300 bp fragments by agarose gel fractionation (3% Nusieve GTG; Lonza/Fisher Scientific) and purified using the QIAquick Gel Extraction Kit (QIAgen).  The libraries contain P7 sequence at the 3’ end downstream of poly(A) and P5 at the 5’ end.

treatment_protocol: Clinical tissue samples were formalin-fixed and paraffin-embedded (FFPE).

tag: tissue id

tag: patient number

tag: disease state

value: normal breast

tag: tissue

value: breast


title: STT5829_Breast_024_EN

source_name: early neoplasia

value: early neoplasia


title: EMSA-Seq of p50p50 round 4 of SELEX

city: Nanjing

first: Jinke

last: Wang

state: Jiangsu

address: Sipailou 2

department: Biomedicine

country: China

institute: Southeast Uiniversity

description: EMSA-Seq selection of ligands that bind a transcription factor

library_source: genomic

data_processing:
Basecalls performed using CASAVA version 1.4
Raw data in the form of reads (FASTQ format) obtained after deep sequencing has been subsequently processed using both UNIX and perl scripts. The main processes included (i)trimming the adaptor sequence from each reads; (ii) matching the sequences of the paired reads to remove the reads with unmatcghed sequences; (iii) checking the definite base calling (A, C,G and T) to remove reads with N; (iv) checking the valid barcode and constant regions to remove reads with invallid barcode and constant regions including incorrect bases and length; (v)checking the unique random sequence at the length of 16 bp to remove reads with incorrect length; (vi)sorting reads according to barcodes: the reads belonging to Round 4 to 0 were ended with AAAA and TTTT, GAAG and GTTT, AGAG and GTCT, AAAG and GTGT, and CAAG and GTGC, respectively; (vii) removing flanking constant regions and barcode sequences from reads and obtained the 16-mer target sequences.
Genome_build: No genome build was perfomed. This item is inapplicable to this study.
Supplementary_files_format_and_content: Fasta files for the final 16-mer sequences obtained for Round 0 to 4.

molecule: genomic DNA

extract_protocol:
For each round of SELEX selection, two protein binding reactions (all 10 μl) were performed simultaneously. Reaction 1 contained 200 ng 64-bp positive dsDNA, 115 ng (Reaction 2) p50 protein (Promega) in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT. Reaction 2 contained 200 ng 64-bp random dsDNA, 115 ng p50 protein in 10 mM Tris-HCl, pH7.5, 50 mM NaCl, 0.5 mM EDTA, 3 mM MgCl2, 1 % glycerol, 0.5 mg/ml BSA, 0.05% NP-40, 0.01 mM DTT and 0.1 μg polydI-dC (Amersham). The protein binding reactions were incubated at room temperature for 60 min and loaded on 6% native polyacrylamide gel and migrated in 0.5 × TBE buffer for 1.5 hs. After electrophoresis, the gel was directly visualized on UV transilluminator and the gel slice containing the shifted dsDNA (Reaction 2) was excised according to the position of the shifted band of Reaction 1. The gel slice was socked in 100 μl diffusion buffer [0.5 M NH4Ac, 10 mM Mg(Ac)2, 1 mM EDTA, pH8.0, 0.1% SDS] overnight at 37°C. The diffusion buffer was purified with Microcon YM-30 and DNA was eluted in 25 μl ddH2O. For experiments in which several rounds of SELEX were carried out the following procedure was adopted. DNA obtained after the first round of TF-DNA binding followed by EMSA, elution and purification was amplified using a high-fidelity PCR procedure. Briefly, multiple 10 μl reactions each comprised of 1 mM dNTPs, 4μM of Primers 1 and 2 (using nested primers: R-F1 and R-R1 for Round 1, R-F2 and R-R2 for Round 2; R-F3 and R-R3 for Round 3; R-F4 and R-R4 for Round 4), together with 1 units of PrimerStar® HS Taq polymerase (TaKaRa) buffered in accompanying 1X PCR Buffer were incubated for 3min at 94°C, then subjected to 20 PCR cycles (1min 94°C, 1min 60°C, 2 min 70°C). DNA was pooled, purified with Microcon YM-30 (Millipore). This was subsequently used as starting material for TF-DNA binding in the second round of SELEX.
The dsDNAs selected in Round 0 to 4 were pooled in equal molar to form a multiplexed sample for sequencing.

tag: selex round

tag: nf-kb dimer


title: STT5830_Breast_024_EN


title: Ecoli_WT_RNAseq

extract_protocol:
Cell culture was rapidly filtered in 250 mL increments at 37 °C over 0.22 μm nitrocellulose ﬁlters (GE) and frozen in liquid nitrogen to simultaneously halt all transcriptional progress. Frozen cells (100 μg) were pulverized on a Qiagen TissueLyser II mixer mill 6 times at 15 Hz for 3 min in the presence of 500 μL frozen lysis buffer (20 mM Tris pH 8, 0.4% Triton X-100, 0.1% NP-40, 100 mM NH4Cl, 50 U/mL SUPERase•In (Ambion)) and 1X protease inhibitor cocktail (Complete, EDTA-free, Roche), supplemented with 10 mM MnCl2. The lysate was resuspended on ice by pipetting. RQ1 DNase I (110 U total, Promega) was added and incubated for 20 min on ice. The reaction was quenched with EDTA (25 mM final), which releases polysomes from the transcript and reduces contamination from ribosomal RNA and ribosome-associated tRNAs. The lysate was clarified at 4 °C by centrifugation at 20,000 g for 10 min. The lysate was loaded onto a PD MiniTrap G-25 column (GE Healthcare) and eluted with lysis buffer supplemented with 1 mM EDTA. Total RNA was purified from the clarified lysate using the miRNeasy kit (Qiagen).
The full library construction protocol was described in detail previously (Churchman et al., Nature 2011). Purified total RNA was fragmented and dephosphorylated with T4 PNK, and then ligated to a 5' adenylated DNA oligo to generate RNA ranging from 30-100 nt. The RNA was reverse transcribed, and the single-stranded DNA circularized and PCR amplified.

value: Total RNA


title: STT5825_Breast_024_IDC

source_name: invasive ductal carcinoma

value: invasive ductal carcinoma


title: STT5763_Breast_022_IDC


title: Ecoli_bicyclomycin_NETseq

description: library strategy: NET-seq

treatment_protocol: For analysis of lacZ transcription, IPTG was added to a final concentration of 1 mM. For bicyclomycin (BCM) treated samples, cells were grown from an initial OD of 0.01 in the absence of BCM. BCM was added to a final concentration of 20 ug/mL when the cells reached OD 0.05.

tag: treated with

value: bicyclomycin (BCM)


title: STT5758_Breast_022_IDC


title: D14_mRNA-seq_replicate_1


title: STT5751_Breast_021_IDC


title: STT6088_Breast_022_EN


title: STT5706_Breast_025_IDC


title: STT5747_Breast_021_EN


title: STT5750_Breast_021_DCIS

source_name: ductal carcinoma in situ

value: ductal carcinoma in situ


title: STT5749_Breast_021_normal


title: STT5669_Breast_020_DCIS


title: STT5670_Breast_020_IDC


title: STT5667_Breast_020_EN


title: STT5828_Breast_024_normal


title: STT5619_Breast_018_EN


title: STT5616_Breast_019_EN


title: STT5618_Breast_018_DCIS


title: STT5614_Breast_019_IDC


title: STT5761_Breast_022_normal


title: STT5756_Breast_022_normal


title: STT5613_Breast_017_DCIS


title: STT5612_Breast_017_EN


title: STT5581_Breast_016_IDC


title: STT5580_Breast_016_EN


title: STT5754_Breast_022_EN


title: STT5578_Breast_015_IDC


title: STT5577_Breast_015_EN


title: STT5576_Breast_015_normal


title: STT5668_Breast_020_normal


title: STT5611_Breast_017_normal


title: STT5574_Breast_014_EN


title: STT5620_Breast_018_normal


title: STT5572_Breast_013_IDC


title: STT5570_Breast_013_normal


title: heat30-5

city: Eugene

first: Patrick

middle: C.

last: Phillips

state: OR

address: 5289 University of Oregon

department: Institute of Ecology and Evolution

institute: University of Oregon

data_processing:
Base-calls were performed using Illumina software
Initial processing was performed using the process_shortreads component of Stacks. In short, reads were demultiplexed, and the barcodes were removed. Reads with ambiguous barcodes were rescued if there were fewer than 2 mismatches, and discarded otherwise. Reads failing the Illumina chastity filter were also removed.
Filtered reads were aligned to the C. remanei genome using GSNAP version 2013-03-31 with parameters -N 1 -s -w 10000 --pairmax-RNA 10000
Reads aligning to genes were counted using htseq-count with parameters -m union -s no
Genome_build: C_remanei-15.0.1; ASM14951v1
Supplementary_files_format_and_content: tab-delimited text files with read counts per gene

organism: Caenorhabditis remanei

source_name: Heat-selected_pooled L1 tissue

growth_protocol: Worms were maintained on NGM-lite seeded with E. coli strain OP50 at 20°C

extract_protocol:
Approximately 100,000 individuals per replicate were flash frozen in Trizol. Total RNA was extracted using a standard Trizol extraction protocol. Poly-A mRNA was isolated from total RNA using the Dynabeads mRNA purification kit (Ambion).
mRNA was sheared to 200-600nt fragments using a buffered zinc solution (RNA fragmentation kit; Ambion). cDNA was synthesized from fragments using Superscript III, and barcoded sequencing adaptors were added to enable multiplexing of samples.

treatment_protocol: Populations were age-synchronized during the embryonic stage via a standard bleaching procedure. Embryos were incubated at either 20°C or 30°C in liquid buffer without food for 20 hours. Pooled L1 larvae were passed through a 20 micron filter immediately prior to tissue collection.

value: Heat-selected, derived from PX443

tag: developmental stage

tag: temperature

tag: batch

value: F


title: heat30-6


title: heat30-3


title: STT5568_Breast_012_DCIS


title: heat30-2

value: E


title: heat30-4


title: STT5567_Breast_012_EN


title: heat30-1


title: STT5511_Breast_011_EN


title: STT5510_Breast_011_normal


title: STT5503_Breast_010_EN


title: heat20-4


title: heat20-6


title: heat20-3


title: STT5502_Breast_010_normal


title: STT5477_Breast_009_IDC


title: heat20-2


title: control30-6

source_name: Control selected_pooled L1 tissue

value: Control selected, derived from PX443

value: D


title: heat20-1


title: STT5476_Breast_009_EN


title: STT5575_Breast_014_IDC


title: STT5571_Breast_013_EN


title: STT5475_Breast_009_normal


title: control30-5


title: STT5474_Breast_008_IDC


title: STT5466_Breast_007_normal


title: STT5573_Breast_014_normal


title: control30-3


title: heat20-5


title: control30-2

value: C


title: STT5504_Breast_010_DCIS


title: STT5465_Breast_007_EN


title: control20-6


title: control20-5


title: STT5451_Breast_006_normal


title: STT5450_Breast_006_DCIS


title: control20-4


title: control20-3


title: STT5463_Breast_007_IDC


title: STT5449_Breast_006_EN


title: control20-2


title: STT5442_Breast_004_EN


title: ancestor30-6

source_name: ancestor_pooled L1 tissue

value: A


title: control20-1


title: ancestor30-5


title: STT5441_Breast_004_normal


title: control30-4


title: STT5472_Breast_008_normal


title: STT5617_Breast_019_normal


title: ancestor30-4

value: B


title: STT5440_Breast_003_EN


title: control30-1


title: STT5439_Breast_003_normal


title: ancestor30-3


title: STT5462_Breast_007_DCIS


title: STT5445_Breast_005_EN


title: ancestor30-2


title: STT5437_Breast_003_DCIS


title: STT5431_Breast_002_EN


title: ancestor20-6


title: ancestor20-5


title: ancestor30-1


title: ancestor20-4


title: ancestor20-3


title: STT5446_Breast_005_normal


title: STT5430_Breast_002_normal


title: STT5425_Breast_001_normal


title: STT5427_Breast_023_normal


title: ancestor20-2


title: STT5426_Breast_023_EN


title: STT5424_Breast_001_EN


title: Trophectoderm 24

status: Public on May 02 2014

city: Davis

first: James

middle: Lee

last: Chitwood

address: 1 Shields Avenue

department: Animal Science

institute: University of California, Davis

data_processing:
Filtering for quality by Galaxy FastQC
RNA-Seq analysis using CLC Genomics Workbench Ver. 6.0
Genome_build: NCBI eca_ref_EquCab2.0 assembly and GenBank annotation
Supplementary_files_format_and_content: .csv containing Gene name, BLAST results, sample RPKM, DESEQ expression statistics (fold change, p-value, etc.)

organism: Equus caballus

source_name: TE

growth_protocol: In vivo-produced D8 blastocysts from superovulated mares.

extract_protocol:
Arcturus Picopure (RNA)
Nugen Ovation V1 (cDNA) followed by TruSeq DNA V1

treatment_protocol: Pronase treatment of blastocyst followed by mechanical isolation of TE tissue and purication of ICM by immunosurgery.

value: Trophectoderm


title: ancestor20-1


title: Inner cell mass 25

source_name: ICM

value: inner cell mass


title: Inner cell mass 15


title: Inner cell mass 22


title: Trophectoderm 23


title: Trophectoderm 16


title: ChIP-seq, 3T3-L1 adipocyte FOSL2 untreated

city: Cincinnati

first: Hee-Woong

last: Lim

state: Ohio

address: 3333 Burnet Ave. MLC 7024

department: Division of Biomedical Informatics

institute: Cincinnati Children's Hospital Medical Center

data_processing:
GRO-seq: First, adapter and poly-A sequence were trimmed off from the raw tags, then remaining tags were converted into FASTA format before alignment. Trimmed tags were aligned to the mouse genome, mm8, using Bowtie with the following options: inputs were in FASTA format (-f), three mismatches were allowed for each tags (-v 3), and only uniquely mapped tags were retained (-m 1). All the alignments were adjusted to 50bp by extending toward 3’-end if their size is shorter than that to make libraries comparable.  For stack-height profiles, all the replicates for each condition were pooled. genomeCoverageBed in bedtools was used first to make bedGraph files for plus and minus strands separately, where minus strand values were converted into negative ones. All the bedGraph files were then converted into bigWig files using bedGraphToBigWig utility.
ChIP-seq: For each chip-seq, all the reads were aligned to mouse genome, mm8, using Bowtie with option (--best --strata --fullref -m 1 -k 1). All the redundant tags were eliminated except one per each genomic position before downstream analysis. Peak calling was done using Homer and 1 rpm cutoff was further applied for peak tag counts within 200bp window.
Genome_build: mm8
Supplementary_files_format_and_content: bigWig files: stack-height profile showing nascent transcripts in 3T3-L1 mature adipocyte at various timepoints after rosi treatment.  Peak files: Peak calling results for ChIP-seq experiments before or after rosi treatment.

growth_protocol: 3T3-L1 cells were obtained from American Type Culture Collection and grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Tissue Culture Biologics), 100 U/ml penicillin and 100 μg/ml treptomycin (Invitrogen). Two days post-confluence differentiation media was added, consisting of growth media with 1 μM dexamethasone, 10 μg/ml human insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (Invitrogen). Cells were differentiated as described previously, by growth in differentiation media for 2 days, followed by growth media with insulin for 2 days, followed by growth media only.

extract_protocol: Cells were crosslinked with 1% formaldehyde for 15 minutes and quenched with 0.125M glycine. Cells were probe sonicated to shear the chromatin. Prepared chromatin was incubated with antibody overnight. Protein A Sepharose beads (GE healthcare) were pre-blocked with BSA, added to the samples, and rotated at room temperature for 5 hours. IPs were washed and incubated at 65°C overnight. Samples were treated with Proteinase K for 2 h at 37°C, then extracted with phenol/chloroform and chloroform, then precipitated for 2 h. Pellets were washed and resuspended in TE. DNA was used for qPCR or to prepare sequencing libraries according to the Illumina protocol.

treatment_protocol: When indicated, mature 3T3-L1 adipocytes were treated with 1μM rosiglitazone (Biomol) dissolved in DMSO.

tag: ChIP

tag: antibody

value: sc-13017x, Santa Cruz

tag: cell line


title: ChIP-seq, 3T3-L1 adipocyte JUND untreated

value: JUND

value: sc-74x, Santa Cruz


title: monocyte

city: Shanghai

first: PIN

last: WANG

address: Xiangyin Road 800

institute: SMMU

data_processing:
Basecalls performed using CASAVA
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: hg19
Supplementary_files_format_and_content: fastq file

source_name: periphery blood cells

growth_protocol: Monocytes were purified from PBMCs using anti-CD14 microbeads (Miltenyi Biotech) and then cultured at 37°C in 24-well plates (5x10^5 cells per well) in 1 mL of RPMI-1640 Medium (PAA) with 10% (vol/vol) FCS (PAA) containing 100 ng/mL human GM-CSF and 20 ng/mL human IL-4 (R&D Systems，Minneapolis, MN). Half medium was replaced by fresh medium with GM-CSF and IL-4 at day 3 and day 5.

extract_protocol:
RNA from monoctes and mature DCs were 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: Immature DCs were matured by 300ng/ml LPS (0111:B4, Sigma) on day 5 and harvested on day 7 for mature DCs.

tag: in vitro culture

value: 7d culture


title: mature_DC

value: 0d culture


title: ChIP-seq, 3T3-L1 adipocyte CEBPb untreated

value: CEBPb

value: sc-150x, Santa Cruz


title: ChIP-seq, 3T3-L1 adipocyte ATF2 untreated

value: sc-6233x, Santa Cruz


title: GSK3beta_ChIPSeq

city: Abu Dhabi

first: Piergiorgio

last: Percipalle

state: Abu Dhabi

address: Saadiyat Campus, Abu Dhabi

department: Biology Department, Science Division

country: United Arab Emirates

institute: New York University Abu Dhabi

data_processing:
For sequencing data alignment and analysis, the current assembly of mouse reference genome is missing the ribosomal rDNA repeats, of which there are approximately 400, located on chromosomes 12, 14 and 15. To compensate for this we utilized the procedure of Zentner et al (2011) and constructed a custom reference sequence that contained a non-masked rDNA repeat, taken from BK000964, which was added to the end of chromosome 12 of the MM9 assembly.
The analysis procedure involved the use of the SOAP2 program to map the reads to the constructed reference genome. Sequences with more than two mismatches were discarded from further analysis. The resulting individual sequences were remapped back to the annotated UCSC MM9 reference sequence which allows for the identification of peaks corresponding to the levels of association of the ChIP target with those loci.
Sequence data was filtered to obtain clean data which was then mapped to the reference genome. Only cleanly mapped sequences were used in further analysis.
The distribution of sequence hits along each chromosome is shown in the WIG file output.

source_name: Mouse Embryonic Fibroblasts

growth_protocol: Mouse embryonic fibroblasts cells were grown in DMEM medium (Gibco), supplemented with 10% foetal bovine serum (Gibco) and a 1% penicillin/streptomycin cocktail

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with a GSK3beta antibody.
5 ng of precipitated DNA was used to prepare sequencing libraries at the Bejing Genome Institute (Hong Kong) using the Illumina HiSeq 2000 platform. Briefly, ChIP DNA was fragmented to 100-500bp by sonication. DNA-end was repaired to overhang a 3’-dA, then adapters were ligated to the end DNA fragments. DNA fragments with proper size (usually 100-300bp, including adaptor sequence) were selected after PCR amplification. Finally we got qualified library for sequencing.

tag: cell type

value: Mouse Embryonic Fibroblasts

tag: passages

tag: strain


title: ChIP-seq, 3T3-L1 adipocyte CEBPa untreated

value: CEBPa

value: sc-61x, Santa Cruz


title: ChIP-seq, 3T3-L1 adipocyte NCOR rosi treated

value: NCOR rosi treated

value: rabbit polyclonal, generated in-lab


title: ChIP-seq, 3T3-L1 adipocyte NCOR untreated

value: NCOR


title: Tal1 ChIP

city: Ottawa

first: Marjorie

last: Brand

state: Ontario

address: 501 Smyth Road

country: Canada

institute: Ottawa Hospital Research Institute

description: Tal1 ChIP with no TSA Treatment

data_processing:
Basecalling was performed with Illumina RTA 1.12.4.2
ChIP-Seq reads were aligned to hg19 genome assembly using BWA 0.6.2 using default parameters.
Reads not mapping to the genome, or mapping more than once to the reference genome were removed for followup analysis.
Duplicate unique reads were removed with samtools version 0.1.18 rmdup tool.
Comparative binding analysis was done by calling peaks with MACS using low stringency in each ChIP and then comparing the read tag density for each corresponding peak (as described in Palii et al. submitted). Peaks were only used to do the comparative analysis between Control and TSA treatment ChIP-Seq.
Genome_build: hg19
Supplementary_files_format_and_content: bigWIG files represent the read coverage of the ChIP-Seq across the genome. The files were generated using the BEDTools suite (version 2.16.2) and UCSC genome browser standalone tools for Unix from the aligned reads output of BWA 0.6.2 using default values.

source_name: Human primary endothelial colony-forming cells

growth_protocol: Human umbilical cord blood from normal full-term deliveries were obtained after informed consent. Light-density mononuclear cell (MNC) fractions were isolated by Ficoll density and resuspended in complete EGM-2 medium (EBM-2 Endothelial Cell basal Medium-2, Lonza, Cat# CC-3156, supplemented with EGM-2 SingleQuot Kit Supplements & Growth Factors, Lonza, Cat# CC-4176)  and 10% fetal bovine serum and plated on to CellBIND 6 well plates at 1x107 cells/well. Individual ECFC colonies were isolated with 0.05% trypsin and expanded in EGM-2 medium. ECFCs at passage 3 were used.

extract_protocol:
Binding of TAL1 transcription factor was measured using a ChIP protocol as previously described (Palii, 2011). Chromatin from 0.5x10^8 cells was fragmented to a size range of 100-300 bases with a Bioruptor (Diagenode) at 4°C. Solubilized chromatin was immunoprecipitated with an antibody against TAL1 (C-21, Santa-Cruz) or an isotype IgG control. Antibody-chromatin complexes were pulled-down using Dynabeads-Protein G, washed and eluted. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol-chloroform.
ChIPed DNA was amplified using the Illumina protocol with modifications described in (Palii, 2011).

treatment_protocol: ECFCs in complete EGM-2 medium at 70% confluency were treated with 100 ng/ml final TSA (cat# 19-138; Upstate Millipore) or a vehicle control as indicated. Cells were washed with PBS prior to harvesting and crosslinked with formaldehyde for 30 min. at RT as previously described (Palii, 2011).

tag: source

value: ECFCs derived from human umbilical cord blood

value: anti-TAL1 (C-21, Santa-Cruz)

tag: treatment


title: Mock ChIP With TSA Treatment

description: Mock ChIP with TSA Treatment

value: isotype IgG control

value: 100 ng/ml final TSA


title: Tal1 ChIP With TSA Treatment

description: Tal1 ChIP with TSA Treatment


title: Mock ChIP

description: Mock ChIP with no TSA Treatment


title: ChIP-seq, 3T3-L1 adipocyte p300 untreated

value: sc-585x, Santa Cruz


title: 3T3-L1 adipocyte input

source_name: 3T3-L1 adipocyte input


title: ChIP-seq, 3T3-L1 adipocyte CBP rosi treated

value: CBP rosi treated

value: sc-369x, Santa Cruz


title: ChIP-seq, 3T3-L1 adipocyte MED1 rosi treated, replicate 2

value: A300-793A, Bethyl


title: ChIP-seq, 3T3-L1 adipocyte MED1 rosi treated, replicate 1


title: ECFC_Mock_ChIP

description: Mock ChIP in ECFC cells

data_processing:
Basecalling was performed with Illumina RTA 1.12.4.2
ChIP-Seq reads were aligned to hg19 genome assembly using MAQ 0.7.1 allowing a maximum of two mistmatches. Unmapped reads were truncated to 31 bp and remapped to the genome. Truncation and remapping were repeated down to 26 read size.
Reads mapping to more than two locations on the genome were removed.
Reads mapping only to one location on the genome were used to call peaks with MACS 1.3.7.1 using a minimum cutoff of 8 tags per enriched region with a p-value threshold for peak significant under 1x10^-5.
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files represent the read coverage of the ChIP-Seq over the genome. The files were generated from unique reads without duplicates from the alignment with MAQ using the UCSC genome browser standalone tools for Unix

extract_protocol:
Binding of TAL1 transcription factor was measured using a ChIP protocol as previously described (Palii, 2011). Chromatin from 0.2x10^8 cells was fragmented to a size range of 100-300 bases with a Bioruptor (Diagenode) at 4°C. Solubilized chromatin was immunoprecipitated with an antibody against TAL1 (C-21, Santa-Cruz) or an isotype IgG control. Antibody-chromatin complexes were pulled-down using Dynabeads-Protein G, washed and eluted. After cross-link reversal and proteinase K treatment, immunoprecipitated DNA was extracted with phenol-chloroform.
ChIPed DNA was amplified using the Illumina protocol with modifications described in (Palii, 2011).


title: ECFC_Tal1_ChIP

description: Tal1 ChIP in ECFC cells

value: against TAL1 (C-21, Santa-Cruz)


title: GROseq, 3T3-L1 adipocyte rosi+3h, replicate 2

description: GRO-seq, nascent RNA

data_processing:
Library strategy: GRO-Seq
GRO-seq: First, adapter and poly-A sequence were trimmed off from the raw tags, then remaining tags were converted into FASTA format before alignment. Trimmed tags were aligned to the mouse genome, mm8, using Bowtie with the following options: inputs were in FASTA format (-f), three mismatches were allowed for each tags (-v 3), and only uniquely mapped tags were retained (-m 1). All the alignments were adjusted to 50bp by extending toward 3’-end if their size is shorter than that to make libraries comparable.  For stack-height profiles, all the replicates for each condition were pooled. genomeCoverageBed in bedtools was used first to make bedGraph files for plus and minus strands separately, where minus strand values were converted into negative ones. All the bedGraph files were then converted into bigWig files using bedGraphToBigWig utility.
ChIP-seq: For each chip-seq, all the reads were aligned to mouse genome, mm8, using Bowtie with option (--best --strata --fullref -m 1 -k 1). All the redundant tags were eliminated except one per each genomic position before downstream analysis. Peak calling was done using Homer and 1 rpm cutoff was further applied for peak tag counts within 200bp window.
Genome_build: mm8
Supplementary_files_format_and_content: bigWig files: stack-height profile showing nascent transcripts in 3T3-L1 mature adipocyte at various timepoints after rosi treatment.  Peak files: Peak calling results for ChIP-seq experiments before or after rosi treatment.

extract_protocol:
GRO-seq: GRO-seq was performed as previously described (Core et al. 2008; Wang et al. 2011). Cells were washed twice with ice-cold PBS, then swelled in swelling buffer (10 mM Tris pH 7.5, 2 mM MgCl2, 3 mM CaCl2) for 5 min on ice. Cells were centrifuged at 400g for 10 min, then resuspended in lysis buffer (swelling buffer with 10% glycerol and 1% Igepal) and incubated on ice for 5 min. Nuclei were washed twice with lysis buffer, then resuspended in freezing buffer (50 mM Tris pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). Nuclei were counted, pelleted, and 5 x 106 nuclei were resuspended in 100 μl freezing buffer. For each library, run-on was performed on 4 tubes of 5 x 106 nuclei.   For the run-on, cells were mixed with an equal volume of run-on buffer (10 mM Tris pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 20 units of SUPERase-In, 1% Sarkosyl, 500 μM ATP, GTP and Br-UTP, 2 μM CTP) and incubated for 5 min at 30°C. Nuclear RNA was extracted with TRIzol (Invitrogen) and precipitated with NaCl and ethanol overnight. The pellet was resuspended in water and the RNA was DNase treated (Ambion) for 30 min. RNA was hydrolyzed using fragmentation reagents (Ambion) for 13 min at 70°C and purified through a Micro Bio-Spin p-30 column (Bio-Rad) according to manufacturer’s instructions. RNA was treated with 1.5μl T4 polynucleotide kinase (New England Biolabs) for 1h at 37°C, then with an additional 1μl for 1h more. RNA was denatured for 5 min at 65°C.   Anti-BrU agarose beads (Santa Cruz) were rotated for 1h in blocking buffer (0.5x SSPE, 1 mM EDTA, 0.05% Tween-20, 0.1% PVP, and 1 mg/ml BSA). Run-on RNA was rotated with beads for 1h, followed by washes twice in binding buffer (0.5x SSPE, 1 mM EDTA, 0.05% Tween-20), twice in low salt buffer (0.2x SSPE, 1 mM EDTA, 0.05% Tween-20), once in high salt buffer (0.5x SSPE, 1 mM EDTA, 0.05% Tween-20, 150 mM NaCl), and twice in TET buffer (TE pH 7.4, 0.05% Tween-20). BrU-labeled RNA was eluted from the beads four times for 15 min with 100 μl elution buffer pre-heated to 42°C. RNA was ethanol-precipitated overnight.
GRO-seq: Precipitated RNA was resuspended in water, denatured, and treated with poly(A)-polymerase (NEB) for 30 min at 37°C. cDNA synthesis was performed as described previously (Wang et al, Nature 2011) using oNTI223 primer ((5′-pGATCGTCGGACTGTAGAACTCT;CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3′) where the p indicates 5′ phosphorylation, ‘;’ indicates the abasic dSpacer furan and VN indicates degenerate nucleotides. The reaction was treated with  3 μl exonuclease I (Fermentas) for 15 min at 37°C, followed by 2 μl 1M NaOH for 20 min at 98°C, and neutralized with 1μl 2M HCl. cDNA was run on 10% TBE-urea gel, then products were excised and eluted from shredded gel pieces for 4h in TE + 0.1% Tween and precipitated in ethanol overnight.   First-strand cDNA was circularized with CircLigase (Epicentre), denatured for 10 min at 80°C, and relinearized with APE I (NEB). Linearized DNA was amplified by PCR using Phusion Hot Start II Kit, according to manufacturer’s instructions. The oligonucleotide primers oNTI200 (5′-CAAGCAGAAGACGGCATA-3′) and oNTI201 (5′-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3′) were used for amplification. The PCR product was run on a 10% TBE gel and eluted as before. Libraries were sequenced on an Illumina hi-Seq2000 with sequencing primer 5′-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3′.


title: ChIP-seq, 3T3-L1 adipocyte MED1 untreated, replicate 1


title: GROseq, 3T3-L1 adipocyte rosi+1h, replicate 2

description: GRO-seq, nascent RNA


title: ChIP-seq, 3T3-L1 adipocyte MED1 untreated, replicate 2


title: GROseq, 3T3-L1 adipocyte rosi+10min, replicate 2

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte rosi+30min, replicate 2

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte rosi+3h, replicate 1

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte untreated, replicate 2

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte rosi+1h, replicate 1

description: GRO-seq, nascent RNA


title: zld_input_replicate2

city: New York

first: Stephen

last: Small

state: NY

address: 100 Washington Square East

department: Biology

institute: New York University

data_processing:
Raw reads were mapped to Drosophila melanogaster genome release 5.3 by Bowtie2 using default settings and duplicated reads were removed on the Galaxy platform.
Peaks were called using the MACS (v.1.4.2 20120305) program with the respective genomic inputs as controls on the Cistrome platform. Parameters used in the MACS program were as follows: effective genome size: 1.52e+08; band width: 300bp; model fold: (10, 30); p-value cutoff 1.00e-05; range for calculating regional lambda: (1000bp, 10000bp).
Genome_build: dm3 (GCA_000001215.2)
Supplementary_files_format_and_content: Peak files were generated by MACS using merged BAM files from two biological replicates; each file summarizes positions, tag number, p value, FDR and enrichment of Bcd-binding peaks.

organism: Drosophila melanogaster

extract_protocol:
Embryos (1-3 hours post egg laying) were collected with embryo wash (0.03% Triton-X100 and 140mM NaCl) and dechorionated in 100% bleach for 2 minutes. For each experiment, six aliquots each of 100ul of embryos were transferred to Eppendorf tubes containing 340ul of buffer A1 (15mM HEPES pH 7.5, 15mM NaCl, 60mM KCl, 4mM MgCl2, 0.5% Triton X-100, 0.5mM DDT, and complete EDTA-free protease inhibitor cocktail (Roche)). To each tube, 37% formaldehyde was added to a final concentration of 1.8%. Embryos were then homogenized with an Eppendorf micropestle and fixed on ice for 15’. Cross-linking was stopped by adding glycine to a final concentration of 0.125M. Homogenates were then centrifuged for 3’ at 3000 rpm in a microcentrifuge at 4°C. Supernatant was discarded and 340 ul buffer A1 was added to each tube. Pellets were suspended by vortexing and centrifuged two more times (three washes with buffer A1 in total), followed by two washes in 340ul buffer A2 (15mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton-X100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, complete EDTA-free protease inhibitor cocktail (Roche)). Pellets were then harvested by centrifuging at 14,000 rpm, snap-frozen in liquid nitrogen, and stored in -80°C for long-term storage. For each ChIP experiment, pellets were combined to yield 60 mg total, and sonicated in 200 ul buffer A2. Sonication was performed 4 times 30’’ on and 30’’ off with a Sonic Dismembrator Model 550 (Fisher Scientific) equipped with a microtip at power setting 3. Affymetrix/USB ChIP Assay Kits were used for immunoprecipitation.
Libraries were made with NEXTflex™ ChIP-Seq Kit (BIOO Scientific, #5143-01) and barcoded with NEXTflex™ ChIP-seq Barcodes (BIOO Scientific, #514120).

tag: genotype/variation

value: zelda mutant

value: embryo

value: 1-3 hours post egg laying

tag: chip antibody


title: ChIP-seq, 3T3-L1 adipocyte p300 rosi treated

value: p300 rosi treated


title: GROseq, 3T3-L1 adipocyte rosi+10min, replicate 1

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte rosi+30min, replicate 1

description: GRO-seq, nascent RNA


title: GROseq, 3T3-L1 adipocyte untreated, replicate 1

description: GRO-seq, nascent RNA


title: zld_input_replicate1

tag: tissue


title: ChIP-seq, 3T3-L1 adipocyte CBP untreated

value: CBP


title: zld_Bcd_replicate1

description: processed data file: Peaks_zld.txt

value: Bcd

tag: chip antibody ref.


title: wt_input_replicate2

value: wild type


title: wt_input_replicate1


title: wt_Bcd_replicate2

description: processed data file: Peaks_wt.txt


title: wt_Bcd_replicate1

description: processed data file: Peaks_wt.txt


title: GA5166

city: baltimore

first: Xin

last: Chen

state: MD

address: 3400 N Charles St

institute: The Joihns Hopkins University

data_processing:
Illumina platform according to its protocol including base call.
ChIP-seq reads were aligned to the dm3 genome assembly using bowtie 0.12.7 with the running parameters (bowtie -p 8 -t -a --phred33-quals -n 2 -e 70 -l 48 -m 1 --best --strata), which allows up to two mismatches for unique match with the fly genomic sequence
Aligned reads are further filtered to remove redundancy.
peaks were called using MACS version 2.0.9 with the default setting
samtools are utilized whenever format conversion is needed.
Genome_build: dm3 (from flybase.org r5.43)
Supplementary_files_format_and_content: wig files were generated using inhouse software pipeline
Supplementary_files_format_and_content: wig files: post procession read density along genome
Supplementary_files_format_and_content: MACSpeaks.wig: peak call
Supplementary_files_format_and_content: counts: average read count per thousand bp on major chromosome arms

source_name: fruitfly testis nos>shRNA E(z)

extract_protocol:
4% formaldehyde fixed testes were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
libraries are prepared following G. Wei, G. Hu, K. Cui, K. Zhao, Methods Enzymol 513, 297 (2012).

value: testis specifically expressing E(z) shmiR RNAi in germ cells

tag: antibody vendor/lot#

value: Millipore, #07-449


title: GA5167


title: zld_Bcd_replicate2

description: processed data file: Peaks_zld.txt

tag: developmental stage


title: GA5169

tag: sample type

value: input DNA


title: Input DNA N6

first: Daniel

middle: A

address: 35 Medical Center Way RMB 933B

department: Neurological Surgery

institute: University of California, San Francisco

description: Input for H3K27me3 (30 hr post differentiation)

data_processing:
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie2 using default configurations
For H3K27me3 Peaks were called using MACS with the following parameters: --no model, --no lamba. Bandwith = 300, Model fold = 10,30, pvalue cutoff = 1.00e-05 as previously described (Feng J et al 2011)
For H3K4me3 Peaks were called using MACS using default parameters
Genome_build: mm9
Supplementary_files_format_and_content: .wig, .txt

source_name: 30 hr differentiation Neural Stem Cells

growth_protocol: SVZ NSC monolayer cultures were established and maintained as previously described (Scheffler et al., 2005).

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared using standard Illumina protocols

value: SVZ derived neural stem cells

value: 5 to 7


title: Input DNA H3K4me3

description: Input for H3K4me3

source_name: Ventral Proliferating neural stem cells


title: Input DNA N5

description: Input for H3K27me3 (proliferating)

source_name: Proliferating Neural Stem Cells


title: H3K27me3_ChIPSeqN6

description: ChIP for H3K27me3 (30 hr post differentiation)

value: H3K27me3 Millipore 07-449


title: H3K27me3_ChIPSeqN5

description: ChIP for H3K27me3 (proliferating)


title: WT_EB_RNASeq

city: Birmingham

first: Hengbin

state: AL

address: 720 20th St S Kaul Bldg 430

department: Biochemistry and Molecular Genetics

institute: University of Alabama at Birmingham

description: RNA

data_processing:
Fastq files for each seq samples are generated by UAB Heflin Genomic Core Facility
After sequencing, the data was converted to FASTQ Sanger format and then aligned to mouse reference genome mm9 by TopHat (Langmead et al., 2009).
Cufflinks was used to assemble transcripts, estimate the abundances, and test for differential expression.
Cuffmerge was used to compare the assembled transcripts to the reference annotation across multiple experiments.
Cuffdiff was then used to determine the change in transcript levels.
tab-delimited text files include RPKM values for each Sample
Genome_build: mm9

source_name: mouse embryoid bodies

growth_protocol: Mouse V6.5 and all derived ESCs were cultured in ESC media, containing DMEM medium (High glucose, Gibco MT-10-013) supplemented with 50 unit/ml penicillin and streptomycin (Life technologies, 15070-063), 18% murine ESCs defined FBS (Thermo Scientific, SH30070.03E), 2mM L-glutamine (Cellgro, 25-005-CI), 1mM sodium pyruvate (Gibco, 11360-070), 1X nonessential amino acids (Cellgro, 25-025-CI, 100X stock), 1X nucleoside (Millipore, ES-008-D, 100X stock), 0.007% β-mercaptoethanol (Fisher, O3446I) and 1000 unit/ml mLIF (Millipore, ESGRO) on irradiated mouse embryonic fibroblasts (Millipore, PMEF-NL) and passaged every other days as described previously (Stock et al., 2007). For induction of Cre recombinase and subsequent Usp16 knock out, Usp162lox:2lox:CAGG Cre ESCs were cultured in medium containing 1μM 4-hydroxytamoxifen (Sigma, H7904 ) for 5 days and then on ESC culture medium. Embryoid bodies (EBs) were formed in EB medium (ESC medium without mLIF) in hanging drops (800 cells per 20μl drop) on petri dish lids for 48 h. EBs were then collected and cultured on a horizontal rotator at 40 rpm in petri dishes and collected at indicated time points. Medium was changed every other day.

extract_protocol:
For ChIP-Seq samples, lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody as elaborated in supplemetal methods.
For RNA-Seq, TRIzol is used for total RNA purification and RNA samples are subjected to protocol as described in supplemental methods.
For ChIP-Seq samples, NEB Next DNA sample prep reagents (New England Biolabs, E6000S/L) were used for end repair, dA-tailing, and adaptor ligation (custom adaptors from Hudson Alpha GSL). The entire volume ligated product was PCR amplified for 15 cycles using Invitrogen's Pfx DNA polymerase using the custom adaptors designed to allow for 96 unique indexes. Size selection was performed on 2% agarose gel. The final library quality was examined by Qubit and Bioanalysis, and real time PCR was performed using KAPA's library quantification kit for Illumina. Each library was diluted to 12.5 pM and clustered using a cBot. Each library was sequenced over 1/6th of a HiSeq 2000 lane following standard Illumina protocols for paired end 50 bp sequencing.
For RNA-Seq samples, cDNA library was constructed with TruSeq library generation kits (Illumina). The cDNA ends were repaired, A-tailed, and adaptor ligated for indexing. The cDNA library was then quantitated by real time PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems) prior to cluster generation. Clusters were then generated to yield approximately 725-825 K clusters/mm2 and the density and quality were determined after the first base addition. Paired end 2 x 50bp sequencing runs were performed for all samples.

treatment_protocol: For induction of Cre recombinase and subsequent Usp16 knock out, ESCs were cultured in medium containing 1μM 4-hydroxytamoxifen (Sigma, H7904 ) for 5 days and then on ESC culture medium.

tag: genotype

value: Usp16 WT

value: mouse embryoid bodies


title: Usp16_KO_EB_RNASeq

description: RNA

value: Usp16 KO


title: Usp16_KO_ESC_RNASeq

description: RNA

source_name: mouse embryonic stem cell

value: mouse embryonic stem cell


title: WT_ESC_RNASeq

description: RNA


title: ubH2A-ChIPSeq_KO_EB

description: ChIP-DNA/genomic DNA

data_processing:
Fastq files for each seq samples are generated by Hudson Alpha Genomic Service Laboratory.
Raw fastq files are groomed and collapse on Galaxy.
The converted fastq files obtained from Hudson Alpha GSL were aligned to mouse reference genome mm9 by BOWTIE on Galaxy public data processing platform.(https://usegalaxy.org)
Aligned fastq files are converted to bed files and used to call peaks by MACS on Galaxy toidentified enriched regions
.bed files generate from fastq for each samples
Genome_build: mm9

value: Millipore, 05-678


title: ubH2A-ChIPSeq_WT_EB

description: ChIP-DNA/genomic DNA


title: H2A-ChIPSeq_WT_EB

description: ChIP-DNA/genomic DNA

value: Millipore, 07-146


title: H2A-ChIPSeq_KO_EB

description: ChIP-DNA/genomic DNA


title: Usp16_ChIPSeq_KO_EB

description: ChIP-DNA/genomic DNA

value: Usp16 purified antibody (Non commercial, available upon request)


title: Usp16_ChIPSeq_WT_EB

description: ChIP-DNA/genomic DNA


title: H2A-ChIPSeq_KO_ES

description: ChIP-DNA/genomic DNA


title: H2A-ChIPSeq_WT_ES

description: ChIP-DNA/genomic DNA


title: ubH2A-ChIPSeq_WT_ES

description: ChIP-DNA/genomic DNA


title: ubH2A-ChIPSeq_KO_ES

description: ChIP-DNA/genomic DNA


title: Usp16_ChIPSeq_KO_ES

description: ChIP-DNA/genomic DNA


title: Usp16_ChIPSeq_WT_ES

description: ChIP-DNA/genomic DNA


title: IgG-ChIPSeq_WT_ES

description: ChIP-DNA/genomic DNA

value: Millipore, AP132


title: H3K4me3_ChIPSeqN5

description: ChIP for H3K4me3

value: H3K4me3 Active Motif 39159


title: FNY01.2_2.Ery.NS.Lexo.round2

status: Public on May 01 2014

city: Bronx

first: Eric

middle: E

last: Bouhassira

address: 1300 Morris PArk Ave

department: Cell Biology

institute: Albert Einstein College Of Medicine

description: sample 4

data_processing:
library strategy: Nascent strand sequencing
Base called using CASAVA
read aligned using bwa
peak calling using MACS 1.42 and Genomic control as base line (see file genomic control 1-4)
Genome_build: hg19
Supplementary_files_format_and_content: Files labeled MAT or PAT contain allele specific data (MAT=maternal chromosome; PAT=paternal chromosome); Files not labelled MAT or PAT contain non-allele-specific data; Genomic control was used to call the peak and was prodcued by sequencing sheared genomic DNA. Files labeled Ery were produced from cultured primary human basophilic erythroblasts.  Files labeled WBC were produced from circulating mononuclear white blood cells.

growth_protocol: Mononuclear cells were isolated from peripheral blood by density gradient centrifugation on Histopaque and were expanded and differentiated into basophilic erythroblasts in culture for two weeks in serum-free condition as described in  PMID 18024790

extract_protocol:
Genomic DNA was extracted using standard procedure
Illumina standard single-end protocol was used. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 10 cycles and library fragments of various sizes (300 to 500 depending on libraries)  were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: Short, newly replicated DNA were extracted using either the lambda exonuclease procedure or using immunoprecipitation with anti-BrdU antibodies

tag: sample source

value: healthy control

value: cultured primary basophilic erythroblasts

tag: nascent strand isolation method

value: lambda exonuclease procedure


title: FNY01.2_2.Ery.NS.BrdU.round2

description: Sample 2

value: immunoprecipitation with anti-BrdU antibodies


title: FNY01.2_2.Ery.NS.Lexo.round1

description: Sample 3


title: FNY01.2_2.Ery.NS.BrdU.round1

description: Sample 1


title: Liver from wildtype (WT) mouse

city: South San Francisco

first: Vikram

last: Agarwal

address: 1130 Veterans Blvd

institute: Calico Life Sciences

description: mRNA

data_processing:
Pooling all biological replicates of a particular sample, differential expression analysis was performed between two samples of interest using cuffdiff v. 2.1.1 (options --library-type fr-firststrand -b mm10.fa -u --max-bundle-frags 100000000) (Trapnell et al., 2013), using mouse transcript models of protein-coding and long non-coding RNA genes annotated in Ensembl release 72.
For each biological replicate, expression level data (measured in Fragments Per Kilobase Million, or FPKM) was computed using cufflinks v. 2.1.1 (options --library-type fr-firststrand -b mm10.fa -u --max-bundle-frags 100000000 --compatible) (Trapnell et al., 2010), using mouse transcript models of protein-coding and long non-coding RNA genes annotated in Ensembl release 72.

source_name: Liver

growth_protocol: Primary hepatocytes were isolated from liver as reported by Zhang et al. (PLoS One, 2012; also see http://www.mouselivercells.com/), grown for 4-6 hours in DMEM 1g/l glucose (1% Penicillin-Streptomycin, 1% Glutamax and 10% fetal bovine serum) and grown for 24 hours in HepatoZYME-SFM (1% Penicillin-Streptomycin and 1% Glutamax) at 300,000 cells per 6-well and lysed 30 hours post isolation.

extract_protocol: For single-end library construction: Total RNA was depleted for rRNA using the Ribo-Zero rRNA Removal Kit (from Epicentre, an Illumina company). RNA libraries were prepared using the dUTP-based, Illumina-compatible NEXTflex Directional RNA-Seq Kit (Bioo Scientific). For paired-end library construction (performed by BGI): Beads with oligo(dT) were used to isolate poly(A) mRNA after total RNA was collected. Fragmentation buffer was added for interrupting mRNA to short fragments (200 to 700nt). Taking these short fragments as templates, random hexamer-primers were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were ligated to sequencing adaptors. For amplification with PCR, fragments were size-selected on an agarose gel. For small RNA-seq libraries (performed by BGI): Total RNA was size fractionated (18-30 nt), followed by 5' and 3' adaptor ligations, RT-PCR, and Solexa sequencing.

treatment_protocol: Adenovirus or Antagomir were added 4-6 hours after isolation and left on the cells for 24 hours

value: whole liver

value: wild type


title: Liver perfused with Insulin

description: mRNA

value: liver perfused with Insulin


title: Liver from mouse fed with high fat diet (HFD)

description: mRNA

value: high fat diet


title: Liver from Ldlr deficient mouse (Ldlr-/-)

description: mRNA

value: Ldlr deficient mouse (Ldlr-/-)


title: Antagomir-122mm

description: mRNA

source_name: Primary hepatocytes

value: cells derived from liver

value: treated with Antagomir-122mm


title: IgG-ChIPSeq_WT_EB

description: ChIP-DNA/genomic DNA

value: Usp16 WT

tag: cell line

value: mouse embryoid bodies


title: Liver perfused with PBS

description: mRNA

value: liver perfused with PBS


title: Liver from mouse fed with chow diet (small RNA)

description: small RNA

data_processing: For small RNA-seq data, we counted the number of occurrences in which the first 20 nucleotides of the raw sequence matched a known mature Mus musculus miRNA sequence deposited in miRBase version 20.

value: chow diet


title: MOI200 Ad-AldoA, three miR-122 sites, rep2

description: mRNA

value: primary hepatocytes expressing the transcript of AldolaseA (Ad-AldoA), containing 3 sites matching miR-122


title: MOI200 Ad-AldoA, three miR-122 sites, rep3

description: mRNA


title: Antagomir-122, rep2

description: mRNA

value: treated with Antagomir-122


title: MOI200 Ad-AldoA, three miR-122 sites, rep1

description: mRNA


title: MOI200 Ad-AldoA, one miR-122 site, rep3

description: mRNA

value: primary hepatocytes expressing the transcript of AldolaseA (Ad-AldoA), containing  1 site matching miR-122


title: MOI200 Ad-AldoA, one miR-122 site, rep2

description: mRNA


title: MOI200 Ad-AldoA, one miR-122 site, rep1

description: mRNA


title: MOI20 Ad-AldoA, three miR-122 sites, rep3

description: mRNA


title: MOI200 Ad-AldoA, no mir-122 site, rep2

description: mRNA

value: primary hepatocytes expressing the transcript of AldolaseA (Ad-AldoA), containing a mutated miR-122 site (no site)


title: MOI20 Ad-AldoA, three miR-122 sites, rep2

description: mRNA


title: MOI200 Ad-AldoA, no mir-122 site, rep1

description: mRNA


title: MOI20 Ad-AldoA, three miR-122 sites, rep1

description: mRNA


title: MOI20 Ad-AldoA, one miR-122 site, rep2

description: mRNA


title: Liver from mouse fed with chow diet

description: mRNA


title: MOI20 Ad-AldoA, no mir-122 site, rep3

description: mRNA


title: MOI20 Ad-AldoA, no mir-122 site, rep2

description: mRNA


title: MOI20 Ad-AldoA, no mir-122 site, rep1

description: mRNA


title: MOI20 Ad-AldoA, one miR-122 site, rep1

description: mRNA


title: MOI2 Ad-AldoA, three miR-122 sites, rep1

description: mRNA


title: MOI2 Ad-AldoA, three miR-122 sites, rep2

description: mRNA


title: Antagomir-122, rep1

description: mRNA


title: MOI2 Ad-AldoA, one miR-122 site, rep3

description: mRNA


title: MOI2 Ad-AldoA, one miR-122 site, rep2

description: mRNA


title: MOI2 Ad-AldoA, one miR-122 site, rep1

description: mRNA


title: MOI2 Ad-AldoA, no mir-122 site, rep1

description: mRNA


title: MOI2 Ad-AldoA, no mir-122 site, rep2

description: mRNA


title: wild

city: Beijing

first: Zechen

last: Chong

address: NO. 7 Beitucheng West Road

institute: Beijing Institute of Genomics

description:
Trrap-CNSD (TrrapF/F NestinCre+) and the wild are actually heterozygous for the mutation (TrrapF/+ NestinCre+).
Sample 1

data_processing:
RNA-seq short reads of 50 bp were mapped to the mouse genome (mm9) using Tophat2
Differential expression analysis was performed by Cufflinks2
Genes with qvalue<0.05 were selected
Genome_build: mm9
Supplementary_files_format_and_content: The significant DEG list file containing the following columns: test_id, gene_id gene, locus, sample_1, sample_2,  status, value_1, value_2, log2(fold_change), test_stat, p_value, q_value, significant.

source_name: WT_embryo cortex

extract_protocol:
RNA was isolated from E14.5 cortices using the RNeasy Lipid Tissue Mini Kit (Qiagen, Venlo, Netherlands). RNA integrity was checked using the Agilent Bioanalyzer 2100.
Around 2.5µg of total RNA was then used for library preparation using TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diago, California, USA) according to manufacturer’s description.

tag: strain background

value: embryo cortex; brain


title: Trrap knockout

description:
Trrap-CNSD (TrrapF/F NestinCre+) and the wild are actually heterozygous for the mutation (TrrapF/+ NestinCre+).
Sample 2

source_name: Trrap KO_embryo cortex

value: Trrap knockout


title: Day4

city: Singapore

first: Paul

last: Robson

address: 60 Biopolis Street, Genome

department: Developmental biology

country: Singapore

institute: Genome Institute of Singapore

data_processing: ABI bioscope software was used to align the reads to hg18 assembly and to produce the processed data .bam files.

source_name: differentiated human embryonic stem cells

growth_protocol: H1 human ES cells were grown feeder free in conditioned medium. The cells were passaged every 7 days with a split ratio of 1: 3 - 6.

extract_protocol: Total RNA was extracted from cells using a combination of the trizol protocol and the RNAeasy mini kit (Qiagen, #74106). Poly A RNA was extracted using the Poly (A) Purist Mag kit (Ambion # AM 1992).  ABI whole transcriptome library preparation kit (#4425680) was used to generate the RNA-Seq libraries.

treatment_protocol: The cells were exposed to the differentiation medium (changed daily) for the duration of differentiation timepoint.

value: ES cell line H1

tag: timepoint


title: Day0

source_name: human embryonic stem cells


title: Day2


title: Day6


title: MEF7

first: Xiannian

last: Zhang

address: Yiheyuan Road No.5

institute: Peking University

description:
MEF 8-cell Chip
Negative control

data_processing:
Illumina Casava1.8 software used for basecalling.
The raw reads were filtered to discard low-quality reads (reads containing more than 50 bases with quality value ≤ 5 and >10% bases as N). The results were clean reads. Adaptors with flanking polyA/T sequences were trimmed. Additionally, reads with AT content larger than 70% were also removed. Trimmed reads with length larger than 30 bp were kept.
Filtered reads were mapped to the mouse reference sequence downloaded from the UCSC table browser with Burrows-Wheeler Aligner (version 0.6.2-r126). The reads that mapped to RefSeq genes were counted and normalized by reads per kilobase transcript per million mapped read (RPKM).
Genome_build: GRCm38 (mm10)
Supplementary_files_format_and_content: Gene symbols and the corresponding RPKM abundance measurements.

source_name: embryonic fibroblast, negative control

extract_protocol:
Cells are lysed and total RNA are mixed with reverse transcription (RT) master mix containing poly(T) primers with anchor sequences.
The library preparation kits (E7370) were purchased from New England Biolabs. Sequencing library construction and template preparation were performed according to the New England Biolabs library preparation protocols.

tag: strain/background

tag: cell type

value: embryonic fibroblast


title: Rice Nipponbare MNase-seq replicate 1 techrep 2

city: Madison

first: Yufeng

last: Wu

state: WI

address: 1575 Linden Drive

institute: UW-Madison

description: MNase-seq.single.end.gff

data_processing:
We mapped the MNase-seq reads to the rice genome (TIGR release 5) using MAQ software (Li et al., 2008a) with default parameters (except 1-bp mismatch allowed). Only the reads aligning to a unique position in the rice genome were used for further analysis. DNase-seq and RNA-seq dataset were generated from our previous work (Zhang et al., 2012b). Methods for mapping DNase-seq and RNA-seq reads were described previously (Zhang et al., 2012b).
Genome_build: TIGR5
Supplementary_files_format_and_content: The positions of unique mapped reads of single-end MNase-seq were shown in gff files. We used NucleR to detect the position of nucleosome based on pair-end data of MNase-seq

organism: Oryza sativa

source_name: seedling

growth_protocol: Rice cultivar “Nipponbare” seeds were germinated at room temperature for three days. Germinated seeds were then sowed in soil to continue to grow in the greenhouse.

extract_protocol:
The nuclei were then digested with a series of concentrations of micrococal nuclease (MNase). The MNase-digested DNA was separated using 2% agarose gel containing ethidium bromide and visualized under UV light. Nuclei were digested into ~80% nucleosome monomers and ~20% dimers. The mono-nucleosomal DNA was then excised from the gel and purified using a gel purification kit (Qiagen, 28006).
The purified DNA was used for MNase-seq library development, including end blunting, adding “A” base to the blunt DNA fragments, ligating “A” tailed DNA fragments with either single-end adapter or pair-end adapter, and enriching ligated DNA fragments by PCR. The final, amplified DNA was purified and sequenced with 36 bp SR (single reads) or PE (paired end) on a Illumina GAII platform.

tag: cultivar

value: Nipponbare

value: seedling leaf

value: MNase


title: MEF6

description: MEF 8-cell Chip

source_name: embryonic fibroblast


title: Day8


title: Rice Nipponbare MNase-seq replicate 1 techrep 1

description: MNase-seq.single.end.gff

tag: treatment


title: MEF8

description:
MEF 8-cell Chip
Negative control


title: MEF5

description: MEF 8-cell Chip


title: MEF4

description: MEF 8-cell Chip


title: MEF2

description: MEF 8-cell Chip


title: MEF3

description: MEF 8-cell Chip


title: MEF1

description: MEF 8-cell Chip


title: mESC62

description:
mESC 8-cell Chip
Discarded

source_name: embryonic stem cell

value: embryonic stem cell


title: mESC60

description: mESC 8-cell Chip


title: mESC61

description:
mESC 8-cell Chip
Discarded


title: mESC63

description: mESC 8-cell Chip


title: mESC58

description:
mESC 8-cell Chip
Negative control

source_name: embryonic stem cell, negative control


title: mESC59

description: mESC 8-cell Chip


title: mESC57

description:
mESC 8-cell Chip
Negative control


title: mESC56

description: mESC 8-cell Chip


title: mESC54

description: mESC 8-cell Chip


title: mESC53

description:
mESC 8-cell Chip
Discarded


title: mESC52

description: mESC 8-cell Chip


title: mESC55

description: mESC 8-cell Chip


title: Rice Nipponbare MNase-seq replicate 2

description: Nuc.pos.bed


title: mESC51

description: mESC 8-cell Chip


title: mESC50

description:
mESC 8-cell Chip
Discarded


title: mESC48

description: mESC 8-cell Chip


title: mESC47

description: mESC 8-cell Chip


title: mESC44

description:
mESC 8-cell Chip
Discarded


title: mESC45

description: mESC 8-cell Chip


title: mESC43

description:
mESC 8-cell Chip
Negative control


title: mESC49

description:
mESC 8-cell Chip
Negative control


title: mESC41

description: mESC 8-cell Chip


title: mESC38

description: mESC 8-cell Chip


title: mESC39

description: mESC 8-cell Chip


title: mESC40

description: mESC 8-cell Chip


title: mESC42

description:
mESC 8-cell Chip
Negative control


title: mESC36

description: mESC 8-cell Chip


title: mESC35

description: mESC 8-cell Chip


title: mESC37

description:
mESC 8-cell Chip
Discarded


title: mESC34

description: mESC 8-cell Chip


title: mESC33

description: mESC 8-cell Chip


title: mESC32

description: mESC 8-cell Chip


title: mESC31

description:
mESC 8-cell Chip
Discarded


title: mESC27

description: mESC 8-cell Chip


title: mESC29

description:
mESC 8-cell Chip
Negative control


title: mESC28

description: mESC 8-cell Chip


title: mESC24

description: mESC 8-cell Chip


title: mESC22

description: mESC 8-cell Chip


title: mESC23

description: mESC 8-cell Chip


title: mESC25

description: mESC 8-cell Chip


title: mESC21

description:
mESC 8-cell Chip
Discarded


title: mESC46

description: mESC 8-cell Chip


title: mESC19

description: mESC 8-cell Chip


title: mESC15

description: mESC 8-cell Chip


title: mESC16

description: mESC 8-cell Chip


title: mESC18

description: mESC 8-cell Chip


title: mESC10

description: mESC 8-cell Chip


title: mESC11

description: mESC 8-cell Chip


title: mESC14

description:
mESC 8-cell Chip
Discarded


title: mESC13

description: mESC 8-cell Chip


title: mESC17

description: mESC 8-cell Chip


title: mESC8

description:
mESC 2-cell Chip
Two-cell device

source_name: embryonic stem cell, two-cell device


title: mESC9

description: mESC 8-cell Chip


title: mESC7

description:
mESC 2-cell Chip
Two-cell device


title: mESC6

description:
mESC 2-cell Chip
Two-cell device


title: mESC12

description: mESC 8-cell Chip


title: mESC5

description:
mESC 2-cell Chip
Two-cell device


title: mESC3

description:
mESC 2-cell Chip
Two-cell device


title: mESC2

description:
mESC 2-cell Chip
Two-cell device


title: mESC26

description: mESC 8-cell Chip


title: mESC30

description: mESC 8-cell Chip


title: B40-3

description: 40pg Chip

source_name: embryonic stem cell, extracted mRNA, 40pg


title: B40-2

description: 40pg Chip


title: mESC20

description: mESC 8-cell Chip


title: B8-14

description: 8pg Chip

source_name: embryonic stem cell, extracted mRNA, 8pg


title: B8-16

description: 8pg Chip


title: B8-13

description: 8pg Chip


title: B8-11

description: 8pg Chip


title: B8-9

description: 8pg Chip


title: B8-5

description: 8pg Chip


title: B8-7

description: 8pg Chip


title: B8-6

description: 8pg Chip


title: B8-2

description: 8pg Chip


title: B8-4

description: 8pg Chip


title: B8-12

description: 8pg Chip


title: B8-3

description: 8pg Chip


title: B2-2

description: 2pg Chip

source_name: embryonic stem cell, extracted mRNA, 2pg


title: B2-4

description: 2pg Chip


title: mESC4

description:
mESC 2-cell Chip
Negative control

source_name: embryonic stem cell, two-cell device, negative control


title: mESC1

description:
mESC 2-cell Chip
Two-cell device


title: B2-1

description: 2pg Chip


title: T100

description: 100ng Tube

source_name: embryonic stem cell, extracted mRNA, 100 ng, tube


title: T40-2

description: 40pg Tube

source_name: embryonic stem cell, extracted mRNA, 40pg, tube


title: T40-1

description: 40pg Tube


title: T8-3

description: 8pg Tube

source_name: embryonic stem cell, extracted mRNA, 8pg, tube


title: B8-15

description: 8pg Chip


title: B40-1

description: 40pg Chip


title: T8-2

description: 8pg Tube


title: T8-1

description: 8pg Tube


title: T0

description:
Tube negative control
Negative control

source_name: tube negative control

value: tube negative control


title: NT_Pool_tRNA

city: Rochester

first: Asha

last: Nair

address: 200 1ST ST SW

institute: Mayo Clinic

data_processing:
Base calling was performed with the Illumina CASAVA 1.9 pipeline
QC was performed on the FASTQ files using RSeQC software (http://rseqc.sourceforge.net/)
The paired end reads were aligned by TopHat 2.0.6
Gene counts were generated using HTseq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html)
genome build: hg19
tab-delimited text files including Raw read counts as well as RPKM values for each gene

source_name: A673 Ewing sarcoma cell line

growth_protocol: A673 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 Units/mL penicillin, and 100µg/mL streptomycin.

extract_protocol:
Total RNA was isolated from A673 Ewing sarcoma cells containing control shNT and RUNX3 sh813 shRNAs.  Gene expression was determined with high throughput RNA sequencing using the TrueSeq method (‘poly A RNA Seq’).
RNA libraries were prepared for sequencing using the True Seq SBS Kit v3 protocol from Illumina

treatment_protocol: RUNX3 Sigma Mission shRNA (“sh813”: NM_004350.2-813s21c1) and a control non-target shRNA (shNT) were purchased from Sigma and transduced into A673 Ewing sarcoma cells with lentiviruses. Lentiviruses were made by transiently transfecting HEK 293T cells with the indicated shRNA construct, pMD-G (VSV-G), and EXQV with Lipofectamine 2000. Virus containing supernatants were collected 72 hours later, filtered, and placed on A673 Ewing sarcoma cells in the presence of 6µg/mL polybrene. Cells were incubated with 0.4µg/mL of puromycin for 6 days to select for populations transduced with the virus. RUNX3 expression was determined by immunoblotting as described above.

tag: shRNA expression

value: 813 RUNX3 shRNA


title: B8-10

description: 8pg Chip


title: 813Pool_tRNA

value: NT shRNA


title: B2-3

description: 2pg Chip


title: B8-8

description: 8pg Chip


title: B8-1

description: 8pg Chip


title: mock1

city: ny

first: mingyan

last: lin

address: 1925 morris park ave

institute: aecom

description: processed data file: Mock_FPKM.txt

data_processing:
Pre-processing of raw sequence reads included demultiplexing with CASAVA 1.8.2
transcript trimming of contaminating sequences with Sequence Cleaner (http://sourceforge.net/projects/seqclean/)
removal of vector sequences with UniVec database (http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html)
ASTQ quality trimming using the FASTX Toolkit (http://cancan.cshl.edu/labmembers/gordon/fastx_toolkit/index.html)
HRiMP2.2.3 was used to align sequence reads to annotated transcripts on the UCSC Reference Genome (build GRCh37/hg19). Quantitative analysis, including the statistical analysis of differentially expressed genes (in the SENCR knockdown experiment) was done with Cufflinks 2.0.2 and Cuffdiff2.
Genome_build: hg19
Supplementary_files_format_and_content: One txt file include FPKM for each sample from SENCR kd experiment and another txt file with averaged FPKMs for 3 replicates of mock samples

source_name: muscle cells_mock

growth_protocol: HCASMC grown with propietary medium from manufacturer (Invitrogen)

extract_protocol:
Total RNA was isolated from human coronary artery smooth muscle cells (HCASMC) under normal growth conditions (for initial RNA-seq profile of lncRNAs) or in HCASMC where SENCR was knocked down using RNeasy extraction kit (Qiagen). Deep RNA sequencing (RNA-seq) was performed on the Illumina Genome Analyzer IIx platform by the University of Rochester Medical Center Genomics Research Center (http://www.urmc.rochester.edu/fgc/) after bioanalyzer quality control was confirmed.
RNA libraries were prepared for sequencing using standard Illumina protocols. Briefly, total RNA were extracted to produce cDNA using DT-T7 primers. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified cDNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: 30 nM dicer substrate RNA to SENCR or control sequence for 3 days before RNA isolation

value: human coronary artery smooth muscle cells (HCASMCs)

tag: genotype/variation

value: normal control


title: MM

city: Guangzhou

first: Yanyuan

last: Lei

state: Guangdong

address: No. 7 Jinying Rd., Tianhe District

institute: Guangdong Academy Agricultural Sciences

data_processing:
Illumina Casava 1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Plutella xylostella transcriptom using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads per kilobase of exon model per million mapped reads (RPKM) were calculated using a protocol from Mortazavi et al., 2008 . In short, The RPKM measure of read density reflects  the molar concentration of a transcript in the starting sample by normalizing for RNA length and for the total read number in the measurement. This facilitates transparent comparison of transcript levels both within and between samples.
Genome_build: Plutella xylostella transcriptome
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

organism: Plutella xylostella

growth_protocol: The P. xylostella Cry1Ac-susceptible strain (MM) and Cry1Ac-resistant strains (MK and GK) to Bt toxin Cry1Ac were maintained on cabbage under the same conditions.

extract_protocol:
Total RNA was isolated from MK, GK and MM P. xylostella samples with Trizol reagent. Briefly, mRNA was purified from each sample with Dynal oligo (dT) beads, respectively, Then fragmented the mRNA by using the RNA fragmentation kit, first and second strand cDNA was synthesized using random hexamer primers, The double-stranded cDNA fragments were further processed by an end repair using T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase (NEB) followed by a single <A> base addition using Klenow 3’to 5’ exo-polymerase, and then ligated with Illumina 's adaptor.Finally, the library products were then subjected to sequencing analysis on the flow cell via Illumina HiSeq™ 2000.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: The MM strain was cultured without exposure to any Bt toxins. Before the sample collected, Cry1Ac-resistant P. xylostella were treated with 750?g/mL Bt toxin Cry1Ac to eliminate the heterozygous individuals. Then the survivors were collected after 48 hours and designed as the resistant sample (MK and GK). Then fourth-instars larvae midgut tissues of MK,GK and MM were collected, respectively.  While Cry1Ac resistance was at least 8,000-fold greater for GK than for MM and >10,000-fold greater for MK than for MM.

tag: phenotype

tag: tissue

value: Midgut

tag: development stage

value: fourth-instar larvae


title: mock3

description: processed data file: Mock_FPKM.txt


title: SENCR_ctr_3

description: processed data file: SENCR_FPKM.txt


title: SENCR_kd_3

description: processed data file: SENCR_FPKM.txt


title: SENCR_ctr_1

description: processed data file: SENCR_FPKM.txt


title: mock2

description: processed data file: Mock_FPKM.txt


title: FNY01_3_3_Ery_round 2_S_Phase

data_processing:
library strategy: TimEX-seq
Base called using CASAVA
read aligned using bwa
normalisation (to total number of reads)
smooth data with Gaussian filter
calculate S to G1 ratio
Genome_build: hg19
Supplementary_files_format_and_content: bed and bedgraph. Bedgraph contain high resolution (non-allele-specific) or allele-specific S to G1 ratio representing the Timing of replication. Files labeled MAT or PAT contain allele specific data (MAT=maternal chromosome; PAT=paternal chromosome); Files not labelled MAT or PAT contain non-allele-specific data; Files labeled Ery were produced from sorted cultured primary human basophilic erythroblasts in S phase.  Files labeled WBC were produced from circulating mononuclear white blood cells (G1 cells).

source_name: FNY01_3_3_erythroblasts_S phase

growth_protocol: Mononuclear cells were isolated from peripheral blood by density gradient centrifugation on Histopaque and were expanded and differentiated into basophilic erythroblasts in culture for two weeks in serum-free condition as described in PMID 18024790

extract_protocol:
Genomic DNA was extracted using standard procedure
Illumina standard pair-end protocol was used.Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 10 cycles and library fragments of various sizes (300 to 500 depending on libraries)  were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: basophilic erythroblasts were stained with propidium iodide for DNA content and cells in the S phase of the cell cycle were sorted

value: Healthy control

value: cultured primary basophilic erythroblasts

tag: cell cycle phase

value: S phase


title: SENCR_kd_2

description: processed data file: SENCR_FPKM.txt


title: SENCR_ctr_2

description: processed data file: SENCR_FPKM.txt


title: FNY01_3_3_Ery_round 3_S_Phase


title: FNY01_3_3_WBC_round 1_G1

source_name: FNY01_3_3_WBC_G1 phase

value: circulating mononuclear white blood cells

value: G1 phase


title: GK


title: FNY01_3_3_WBC_round 2_G1


title: FNY01_3_2_WBC_round 2_G1

source_name: FNY01_3_2_WBC_G1 phase


title: MK


title: FNY01_3_3_Ery_round 1_S_Phase


title: FNY01_3_2_WBC_round 1_G1


title: T40-3

description: 40pg Tube


title: FNY01_2_2_WBC_Genomic_control_round2_300bp

description: sample 6

value: healthy control


title: FNY01_3_2_Ery_round 1_S_Phase

source_name: FNY01_3_2_erythroblasts_S phase


title: FNY01_3_2_Ery_round 3_S_Phase


title: FNY01_3_2_Ery_round 2_S_Phase


title: FNY01_2_2_WBC_Genomic_control_round1_300bp

description: sample 5


title: SENCR_kd_1

description: processed data file: SENCR_FPKM.txt


title: nls-Cherry #2

city: Oakland

first: Ravi

last: Sachidanandam

address: 2945 Webster St

institute: Girihlet Inc.

data_processing:
Illumina CASAVA version 1.7 used for basecalling (?)
Mapped to the Danio rerio genome build Zv9/danRer7
Genome annotation from UCSC was used to identify exon expression and expression values for each gene were assigned by normalizing to the median of the coverage at each position of genes with non-zero expression
Values were quantile-normalized allowing comparisons of the two sets using custom-built software and algorithms as described (Aravin et al., 2008; Olson et al., 2008; Tam et al., 2008)
Genome_build: Zv9/danRer7
Supplementary_files_format_and_content: Tab-delimted text file includes reads and their frequencies.

organism: Danio rerio

source_name: Zebrafish 5 dpf liver

extract_protocol: Total small RNA from livers of 5 dpf larval zebrafish were collected: 2 batches of Tg(fabp10:nls-mCherry) control larvae, 2 batches of ethanol-treated Tg(fabp10:nls-mCherry) larvae, and 1 batch of Tg(fabp10:nAtf6-cherry; cmlc2:GFP). Each batch was purified for preparation of high-throughput sequencing libraries.

tag: tissue type

value: liver

tag: developmental stage

value: 5 dpf


title: nls-Cherry #1

tag: genotype


title: nls-Cherry Ethanol #2


title: SCS-rep1

city: Lincoln

first: Chi

state: NE

address: 1901 Vine Street

institute: University of Nebraska - Lincoln

data_processing:
Illumina software used for basecalling.
reads were mapped to genome using Bowtie with up to two mismatch
Reads counts for each gene with trimmed mean of M values (TMM) normalization method. Robinson and Oshlack Genome Biology 2010.
Genome_build: Sbicolor_79
Supplementary_files_format_and_content: Tab-delimited plain text files: the first column is gene IDs and the second column is normalized reads counts

organism: Sorghum bicolor

source_name: root

growth_protocol: 16/8 h photoperiod at 25 °C (day) and 18 °C (night)

extract_protocol: Illumina RNAseq library protocol

value: root

value: SCS


title: High Bulk-rep1

value: High Bulk


title: KS78-rep2


title: SCS-rep3


title: KS78-rep3


title: KS78-rep4


title: SCS-rep2


title: TX623-rep3


title: TX623-rep4


title: nls-Cherry Ethanol #1


title: High Bulk-rep4


title: High Bulk-rep2


title: KS78-rep1


title: Low Bulk-rep3

value: Low Bulk


title: Ch17-rep4


title: TX623-rep2


title: Ch17-rep3


title: Low Bulk-rep1


title: Ch17-rep2


title: High Bulk-rep3


title: TX623-rep1


title: Low Bulk-rep4


title: Low Bulk-rep2


title: Ch17-rep1


title: CK60-rep4


title: SCS-rep4


title: CK60-rep2


title: CK60-rep1


title: CK60-rep3


title: TBS-seq for 0.5ng repeat2

city: Shenzhen

first: Desheng

last: Gong

address: No.7 PengFei road

institute: Agricultural Genomes Institute at Shenzhen

description: tagmentation-based whole genome bisulfite sequencing

data_processing:
After sequencing, adapters were first removed using cutadapt-1.2.1 software (http://code.google.com/p/cutadapt/), only reads with minimally 45 bp length were remained. After removing adapters, low quality reads that contained more than 10% N’s or more than 50% of the sequence with low quality value (quality value <5) per read were filtered out. Read mapping was done on the rice reference genome (IRGSP_build5) using BS-MAP (version 2.73) alignment software that combines genome hashing and bitwise masking to achieve fast and accurate bisulfite mapping, allowing for maximally 5 mismatches or 1 small insertion or deletion.
After mapping, PCR duplicates were removed using Samtools software. Then, we separated the reads into two categories, depending on whether they are from the top (C changed to T, C-T) and bottom (G changed to A, G-A) strands. By simulating the methylation proportion across each possible read position, the 12 bps from the 3’ ends of C-T reads and the 5’ ends of G-A reads were then removed as which were affected by the 5-methyl-dCTPs incorporated during the gap-repair and strand displacement step.
Methylation level was defined as the fraction of read counts of ‘C’ in the total read counts of both ‘C’ and ‘T’ for each covered C site, and herein average percentage methylation of all cytosine residues for any genomic region covered was computed as the fraction of read counts of ‘C’ in the total read counts of both ‘C’ and ‘T’ for each genomic region. Based on such read fraction, methylated cytosines were called using a binomial distribution as in the method described by Lister et al. (2009), whereby a probability mass function is calculated for each methylation context (CpG, CHG, CHH). The non-conversion rate and sequencing error rate were taken into account, for which the total error rate was determined by unmethylated lambda DNA spike-ins
We comprehensively assessed the repeatability, sensitivity as well as specificity and characterized both global and genic methylation patterns of a rice strain by this new T-WGBS, proving its consistency in comparison with LC-WGBS.
Genome_build: IRGSP_build5
Supplementary_files_format_and_content: *.cout.gz files contain 8 columns. The format is that: (1)Chromosome ID, (2)Cytosine position, (3)+/- Watson/Crick chain, (4)Type (CG/CHH/CHG), (5)Base pattern, (6)Methylation Ratio, (7)Methylated reads num, (8)Un-methylated reads num.

source_name: rice leaf

extract_protocol:
Genomic DNA prepared from a rice strain was quantified by QuantiT dsDNA HS assay kit (0.2-100ng; Invitrogen, Q32854, Carlsbad, CA, USA).
10ng ( each aliquot was spiked with 10pg of unmethylated lambda DNA), 1ng and 0.5ng aliquots of genomic DNA were prepared by gradient dilution and then subjected to fragmentation reaction (regardless of the quantity of DNA used ) by incubating with 4 µL Nextera HMW Buffer (Epicentre-Illumina), nuclease-free water (Ambion) to 16 uL and 4 µL prepared Tn5mC transposomes for 12 min at 55°C followed by purification using 36 µl (1.8X) Agencourt AMPure XP magnetic beads with elution in 14 µL EB buffer. An extension step was then performed by adding 2 µL of 10X Thermopol reation buffer (NEB), 2 µL 10mM 5-Methylcytosine dNTP Mix (Zymo Research, D1030), 1 µl of Bst DNA Polymerase Large Fragment (NEB, M0275s) to each reaction and incubating for 20 min at 65°C. Each reaction was spiked with 200ng of sonicated unmethylated lambda DNA (200-400 bp) and then subjected to bisulfite conversion using a Zymo EZ DNA Methylation Kit (D5100) according to recommended protocols with a 14h 50°C dark incubation and 22 µL (2 x11 µL) elution. The purified DNAs were then amplified using 25 µL Kapa 2G Robust Hot Start Ready Mix (Kapa Biosystems), 1 µL 50X Nextera Primer Cocktail (Illumina - compatible) and 1 µL barcoded Illumina-compatible Adaptor 2 (8-bp index) on the thermocycler with the following parameters: 1 min at 95°C; (20 sec at 95°C; 30 sec at 60°C; 45 sec at 72°C) X (10-15) cycles. The prepared libraries were analyzed by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by qPCR and then used for cluster generation and pair-end sequencing with 90 bp reads (PE90) on Illumina Hiseq 2000 using the following sequencing primers: read 1, 5’-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGA-3’; read 2, 5’-GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGA-3; and index read, 5’-TCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3.  We generated two chemistry ligation based WGBS libraries from 1000 ng of genomic DNA prepared from Rice (each library was spiked with 1ng of unmethylated lambda DNA) according to methods described previously [10]. Libraries were sequenced in a strategy with pair-end 90 bp read (PE90) on Illumina Hiseq 2000 according to the standard Illumina protocol.

value: leaf

tag: input(ng)


title: TBS-seq for 1ng repeat2