title: Input_ChIP-Seq_atx

data_processing:
Base calling  was performed following the standard Illumina protocol.
Sequenced ChIP-seq reads were trimmed for adaptor sequence and low-quality sequence,then aligned to the Arabidopsis genome TAIR10 using Bowtie version 1.0.1 with no more than 2 mismatches.
Peaks were called using MACS version 1.4.2 with the following setting: bandwidth(150nt), m-fold(5-50), p-value cutoff(1e-05).
Sequenced RNA-seq reads were trimmed for adaptor sequence and low-quality sequence ,then using Tophat version 2.0.10 and cufflinks version 2.2.1 to identify differentially expressed genes.
Wig files were normalized into the same level by dividing the total counts of tags.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bed files and wig files were generated by MACS version 1.4.2 and wig files were normalized into the same level by dividing the total counts of tags.

source_name: Leaf

extract_protocol:
Nuclei was extracted using a rapid method as described in He et al., 2013. RNA was extracted using an RNeasy plant mini kit following manufacturer's recommendation (Qiagen, http://www.qiagen.com/).
ChIP-seq libraries were constructed using the ChIP-seq DNA Sample Prep Kit (Illumina) according to the manufacturer’s protocol. RNA-seq library construction was performed with NEB Next® mRNA Library Prep Master Mix Set (NEB, Ipswich, MA, USA) following manufacturer’s instructions.

tag: antibody


title: Input_ChIP-Seq_sdg2

status: Public on Jun 07 2017

type: SRA

city: Beijing

first: Yan

last: He

state: Beijing

address: 2 Yuanmingyuan West Road

department: Plant Genetics and Breeding

country: China

institute: China Agricultural University

library_source: genomic

molecule: genomic DNA

organism: Arabidopsis thaliana

growth_protocol: Plants were grown under normal condition with a photoperiod of 16 h light at 22°C and 8h dark at 20°C.

tag: ecotype

value: Columbia (Col-0)

tag: genotype/variation

value: sdg2 mutant

tag: tissue

value: rosette leaf

tag: developmental stage

value: four-week-old


title: H3K4me3_ChIP-Seq_sdg2


title: H3K4me3_ChIP-Seq_atx

value: atx3/4/5 mutant


title: H3K4me2_ChIP-Seq_atx


title: H3K4me3_ChIP-Seq_WT

value: wildtype


title: CC2_12

city: Rochester

first: John

last: Mariani

state: NY

address: 601 Elmwood Avenue

department: Neurology

country: USA

institute: University of Rochester

library_source: transcriptomic

data_processing:
Basecalls performed using CASAVA version 1.8
Reads were aligned using STAR 2.4 with paramters --outFilterMatchNmin = 10 to mouse genome GRCm38.p3
Reads were quantitated with HTSeq-Count using paramater intersection-strict
Differential expression was carried out in R. Genes that were not expressed or the result of technical noise were removed if all four conditions had an additive value of 2 counts per million (CPM) or less.  using the biomaRt package (Durinck, Spellman, Birney, & Huber, 2009). Filtered raw counts were log2 transformed and assigned precision weights by Voom and fed to Limma for differential gene expression analysis.  The TREAT function was used to determine significant differential expression wtih a log2 fold change of 0.5 for Cntf induction, and 0.15 for both Stat3 silencing and interaction.
Genes with an adjusted p-value < 0.05 were deemend significant and genes with an average of 10 transcripts per million (TPM) in any group were maintained.
Genome_build: GRCm38.p3
Supplementary_files_format_and_content: .csv contains aggregated gene counts from HTSeq-Count
Supplementary_files_format_and_content: .xlsx contains significant genes for all three comparisons, their ensembl gene ids, log2 foldchange, adj.P.Val, external gene name, gene biotype, and description

molecule: polyA RNA

organism: Mus musculus

growth_protocol: Oligodendrocyte progenitor cells (OLP) were plated onto poly-D-lysine coated confocal dishes (Mat-Tek, Ashland, MA) in Sato media with the proliferative factors PDGF-AA (10 ng/ml) and bFGF (20 ng/ml). OLP were cultured in a humidified incubator at 5% CO2 and 37oC, with a half change of media every other day.

extract_protocol:
Cell lysate was harvested and RNA was extracted using Qiagen RNeasy Mini kits.
RNA libraries were prepared using TruSeq RNA library prep kits V2.

treatment_protocol: For targeted gene inactivation of floxed genes in vitro, Stat3fl/fl mouse OLP were trypsinized, counted, and plated on 6-well plates (Falcon, Tewksbury, MA, USA) in Sato media with proliferative factors overnight. The following day, either Ad5CMV-eGFP, for empty vector controls, or Ad5CMVCre-eGFP (Gene Vector Core, University of Iowa, IA, USA), to initiate Cre recombination (m.o.i. = 22.5 based on previous night’s counting), was added to media. After four hours of incubation, cells were washed once in EBSS and incubated for 48 hours in Sato media supplemented with proliferative factors.  Control and silenced cultures were washed once with EBSS and treated with either Sato supplemented with vehicle or 100ng/μl Cntf for 2h.

tag: strain background

value: backcrossed onto C57BL/6

value: Stat3 flox/flox

tag: cell type

value: Primary Cortical Oligodendrocyte Progenitor (OLP) cells

tag: viral treatment

tag: treated with

value: 100ng/ul CNTF


title: CC2_11


title: GFPplusRNA1

status: Public on Jun 08 2017

city: Worcester

first: Nathan

middle: D.

last: Lawson

state: MA

address: 364 Plantation Street

department: Molecular, Cell, and Cancer Biology

institute: University of Massachusetts Medical School

data_processing:
MiSeq Reporter was used for base calls and to generate fastq files
Reads from fastq files were mapped to Zv9 using Tophat2 (v2.0.9; Galaxy version 0.6) with default settings
Differentially expressed genes were identified by mapping reads onto a customized ENSEMBL transcript annotation of Zv9 using Cuffdiff (v2.1.1; Galaxy version 0.0.7) with the default settings
Genome_build: Zv9
Supplementary_files_format_and_content: Y1differentialGenes.tab is in tab-delimited format and contains differential gene output from Cuffdiff.  Includes FPKM, log2 fold change, and statistical values for each gene

molecule: total RNA

organism: Danio rerio

source_name: GFPplusRNA

growth_protocol: Tg(fli1a:egfp) embryos were grown to 24 hours post fertiilization

extract_protocol:
Tg(fli1a:egfp) were dissociated, fixed and subjected to fluorescence activated cell sorting, followed by RNA isolation according to the MARIS protcol
Total Script (Epicentre)

tag: strain

value: 24 hours post fertilization

value: endothelial cells


title: CV2_12

value: Vehicle


title: EC2_6


title: CV2_6


title: EC2_12


title: CV2_11


title: CC2_5


title: EV2_11


title: EV2_6


title: EC2_11


title: Stat3 Input2

data_processing:
Basecalls performed using CASAVA version 1.8
ChIP-seq reads were aligned to the GRCm38.p3 genome assembly using Bowtie2 with default settings
Peaks were called using MACS2 against merged input samples using a p-value cutoff of 0.001
Reproducibility of biological samples were analyzed via irreproducible discovery rate (IDR) keeping peaks with an IDR score < 0.05
Blacklisted peaks, as compiled by the ENCODE consortium, were removed and significant IDR peaks were annotated using ChIPpeakAnno.  Peaks falling 10kb upstream or 5kb downstream of transcript TSSs were considered for comparisons to RNA-Seq data.
Genome_build: GRCm38.p3
Supplementary_files_format_and_content: .xls contains peaks called as significant by MACS2 compared to a merged input.
Supplementary_files_format_and_content: .csv file contains significant IDR peaks, their start and end locations, summits, pileups, fold change enrichments, adjusted p-values, transcript strands, relative peak positionand distance to TSS, transcript start and end sites, ensembl gene id, external gene id, and gene description.

source_name: OLP_Stat3 Input

extract_protocol:
Formalin (Sigma) was added to a final concentration of 1% for 10m to crosslink DNA and attached proteins. Crosslinking was halted by addition of 2.5M Glycine in water to a final concentration of .125M and left to incubated at room temperature for 5m. Dishes were then washed twice in PBS, scraped, and pelleted by centrifugation at 1,200 RPM for 5m at 4oC. An extra dish was trypsinized and counted to determine OPC number per plate. Media was removed by suction and pellets were resuspended in cell lysis buffer (85mM KCl, 0.5% NP40, in 5mM HEPES, pH 8) with 200X protease inhibitor cocktail (Cell Signaling) and PMSF (Sigma) in isopropyl alcohol to a final concentration of 1mM being added fresh. Lysed cells were pooled together to a calculated density of 60 million cells in 600μl cell lysis buffer. Lysed cells were then sonicated at 4oC in a Bioruptor plus (Diagenode, Denville, NJ, USA) for 25 cycles (30s on, 30s rest) on high. Resulting suspension was then centrifuged at 14,000RPM at room temperature for 15m to pellet cell debris. Supernatants were transferred to 1.5ml tubes and 3μl of each were removed for DNA extraction (SimpleChIP Chromatin IP Kit, Cell Signaling). Extracted DNA was assessed for fragment size on an Agilent 2100 Bioanalyzer and DNA concentration on a QUBIT 3.0 Fluorometer (Life Technologies, Norwalk, CT, USA). Upon confirmation of sufficient fragment distribution (>10% between 130 and 230bp), 1% of volume from each tube was taken and saved as input. Supernatants were aliquoted proportionally to 30 million cells per tube and anti-Stat3(y705) (Cell Signaling, 1:100) or normal rabbit IgG (Cell Signaling 1:100) were added to each. Tubes were incubated overnight at 4oC with rotation. The next day 30μl of ChIP Grade Protein G Magnetic Beads (Cell Signaling) was added to each reaction for 2h at 4oC with rotation. Washing of immunoprecipated chromatin, elution of chromatin, and DNA purification of immunoprecipitated samples and inputs using spin columns were carried out according to manufacturer’s protocol (SimpleChIP Chromatin IP Kit, Cell Signaling).
10ng of each ChIP and input sample were then used for library construction for Illumina sequencing following manufacturer’s protocol (NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina, NEBNext Multiplex Oligos for Illumina (Index Primers Set 1), New England BioLabs, Ipswich, MA, USA).

treatment_protocol: OLP were treated with 100ng/ml of CNTF (Peprotech) for 15 minutes.

value: 100ng/ml of CNTF (Peprotech) for 15 m

tag: chip antibody


title: EV2_12


title: Stat3 Input1


title: Stat3 ChIP2

source_name: OLP_Stat3 ChIP

value: p-STAT3 (Y705) (Cell Signaling D3A7 Rb mAb 9145S Lot 26)


title: Input_ChIP-Seq_WT


title: H3K4me2_ChIP-Seq_WT


title: Testes_RNAseq_rep2

city: Haining

first: Wanlu

last: Liu

state: Zhejiang

address: 718 East Haizhou Rd.,

department: Zhejiang University - University of Edinburgh Institute

institute: Zhejiang University

description:
RNA (polyA enriched)
Testes_RNAseq

data_processing:
Raw reads in qseq format obtained from sequencer were first converted to fastq format with customized perl script. Reads quality were controlled with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). High quality reads were then aligned to Rhesus macaque Genome (MacaM)(Zimin et al., 2014) or Homo sapiens genome (hg19) reference genome using Tophat(Trapnell, Pachter, & Salzberg, 2009) (v 2.0.13) by using ‘-no-coverage-search’ option, allowing up to two mismatches and only keeping reads that mapped to one location. Basically, reads were first mapped to MacaM or hg19 gene annotation with known splice junction. When reads did not mapped to the annotated genes, the reads were mapped to entire MacaM or hg19 genome. Number of reads mapping to genes were calculated by HTseq (Anders, Pyl, & Huber, 2015) (v 0.5.4) with default parameters. Expression levels were determined by RPKM (reads per kilobase of exons per million aligned reads) in R using customized scripts.  For RNAseq of human PGC and ESC, published datasets (GSM1466233, GSM1466234, GSM1574595, GSM1574596(Irie et al., 2015)) were obtained from GEO and then processed exactly the same as described above.
Genome_build: MacaM
Supplementary_files_format_and_content: RPKM

organism: Macaca mulatta

source_name: Testes

extract_protocol:
RNA was extracted using QIAGEN RNeasy micro kit (catalog number: 74004) according to manufacturer's instructions
RNA-Seq  cKIT sorted rhesus PGCs, rhesus liver cells, and rhesus iPS cells were lysed in 350ul RLT buffer (Qiagen) and RNA was extracted using the RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. RNA was amplified and converted to cDNA using the Ovation RNA-Seq System V2 (Nugen). cDNA was sonicated to DNA fragments within the 200bp range using a Covaris S2 sonicator according to manufacturers instructions (Duty cycle 10%, Intensity 5, Cycles per burst 200, and time 180 sec). Subsequently libraries were generated using the Encore Rapid Library System according to manufacturers instructions. Library concentration was estimated using a Kapa library quantification kit (Cat# 4827) according to manufacturers instructions.  Rhesus Macaque PGC and embryonic liver libraries were run using 100bp paired-end reads on the HiSeq 2000 System (Illumina). The rhesus iPSC libraries were run using 50bp single end read.  ERCC RNA Spike-In Mix (Cat# 4456740, Invitrogen) was used according to manufacturer’s instructions in the liver cells and iPSC samples.

value: Testes

tag: library type

value: Ovation RNASeq System V2

tag: passage number


title: Testes_RNAseq_rep3

description:
RNA (polyA enriched)
Testes_RNAseq


title: Liver_RNAseq_rep4

description:
RNA (polyA enriched)
Liver_RNAseq

source_name: Liver

value: Liver


title: RNA-Seq_atx-rep1

description: processed data file: atxvsWT_gene_exp_diff.txt

data_processing:
Base calling  was performed following the standard Illumina protocol.
Sequenced ChIP-seq reads were trimmed for adaptor sequence and low-quality sequence,then aligned to the Arabidopsis genome TAIR10 using Bowtie version 1.0.1 with no more than 2 mismatches.
Peaks were called using MACS version 1.4.2 with the following setting: bandwidth(150nt), m-fold(5-50), p-value cutoff(1e-05).
Sequenced RNA-seq reads were trimmed for adaptor sequence and low-quality sequence ,then using Tophat version 2.0.10 and cufflinks version 2.2.1 to identify differentially expressed genes.
Wig files were normalized into the same level by dividing the total counts of tags.
Genome_build: TAIR10
Supplementary_files_format_and_content: FPKM values for mutant and wild-type.

source_name: atx3/4/5 mutant_leaf_RNA-seq

extract_protocol:
Nuclei was extracted using a rapid method as described in He et al., 2013. RNA was extracted using an RNeasy plant mini kit following manufacturer's recommendation (Qiagen, http://www.qiagen.com/).
RNA-seq library construction was performed with NEB Next® mRNA Library Prep Master Mix Set (NEB, Ipswich, MA, USA) following manufacturer’s instructions.

value: Columbia (Col-0)


title: Liver_RNAseq_rep3

description:
RNA (polyA enriched)
Liver_RNAseq


title: Liver_RNAseq_rep2

description:
RNA (polyA enriched)
Liver_RNAseq


title: Δhlx2 mutant 3

city: Durham

first: Cynthia

last: Darnell

state: North Carolina

address: 130 Science Drive

department: Biology

institute: Duke University

description: rRNA depleted total RNA

data_processing:
Basecalls performed using
Adapter sequences trimmed using Trim_galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)
Reads aligned using Bowtie2 (Langmead B, Salzberg S. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012, 9:357-359.)
Alignments were converted to .bam files and indexed using sam_tools (Li H A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011 Nov 1;27(21):2987-93. Epub 2011 Sep 8.)
Gene hits were determined using HT-seq package for Python (Simon Anders, Paul Theodor Pyl, Wolfgang Huber. HTSeq — A Python framework to work with high-throughput sequencing data  Bioinformatics (2014))
Genome_build: Bowtie2 based on Halobacterium salinarum NRC-1 (RefSeq NC_002607.1; NC_002608.2; NC_001869.1)
Supplementary_files_format_and_content: .bam files contain aligned reads. .count files contain counts assigned to genes.

organism: Halobacterium salinarum NRC-1

source_name: whole cells

growth_protocol: Cells were harvested during exponential phase (OD600~0.2) during growth 50 ml Complete Media (CM) containing 50 μg/ml uracil in 125 ml unbaffled flasks at 42°C, shaking at 225 rpm

extract_protocol:
Total RNA was extracted using Absolutely RNA Miniprep kit (Agilent) per manufacturer's instructions
RNA was rRNA depeleted using Ribo-Zero kit (Illumina) per manufacturer's instructions. Depleted RNA was converted to cDNA libraries using Stranded RNA library preparation kit from Kapa Biosystems per manufacturer's instructions including 10 rounds of amplification and ligation to Illumina TruSeq adapters 1-6. Quality was determined using a High Sensitivity DNA chip on a Bioanalyzer 2100 (Agilent), pooled, and run on a single lane in an Illumina HiSeq 2500

treatment_protocol: Cells were snap frozen and stored at -80°C overnight before RNA extraction

tag: genotype

value: delta hlx2

tag: biological replicate


title: Ovaries_RNAseq_rep1

description:
RNA (polyA enriched)
Ovaries_RNAseq

source_name: Ovaries

value: Ovaries


title: Δhlx2 mutant 2

description: rRNA depleted total RNA


title: Δhlx2 mutant 1

description: rRNA depleted total RNA


title: Δura3 control 2

description: rRNA depleted total RNA

value: delta ura3


title: riPSC_RNAseq_rep1

description:
RNA (polyA enriched)
riPSC89P13_100K_RNAseq

source_name: riPSC

value: riPSC

value: Passage 13


title: riPSC_RNAseq_rep2

value: Passage 21


title: Input control sample

first: Cuihua

address: Beijing

institute: Institute of Microbiology, Chinese Academy of Sciences

data_processing:
Sequence on Illumina HiSeq 2500
Binding sites were identified using MACS1.4
Peak finding and downstream data analysis were performed using HOMER
Genome_build: Human NCBI genome build 37
Supplementary_files_format_and_content: FASTQ

organism: Homo sapiens

source_name: Input control ChIP in U937 Cells

extract_protocol:
U937 cells stablely expressing Flag-PtpA were seeded at 1×107 cells per 10 cm dish. Cells were crosslinked with 1% formaldehyde for 10 min. Glycine was then added for 5 min to a final concentration of 0.125 M, cells scrapped and collected. The pellet was resuspended in lysis buffer (5 mM Tris-HCl, pH 8.0, 85 mM KCl, 0.5% NP40 supplemented with protease inhibitors cocktail) and subjected to centrifugation to collect nuclear pellet.
The pellet was resuspended in RIPA buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-deoxycholate, 0.1% SDS supplemented with protease inhibitors cocktail). The nuclear extracts were sonicated. The chromatin was incubated overnight with anti-Flag antibody (Sigma) followed by four washes with LiCl wash buffer (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1% NP40, 1% Na-deoxycholate) and 1 wash with TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0). DNA was eluted in Elution Buffer (1% SDS, 0.1 M NaHCO3) for 1 h at 37℃ and used for sequencing library preparation.

tag: cell line


title: T

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

country: France

institute: IPMC/CNRS

data_processing:
Reads were first filtered to remove SOLiD adaptors, SOLiD barcodes and rRNAs reads.
Alignment was performed within Lifescope 2.5.1 software RNAseq pipeline using default parameters.
Raw sequence reads were counts and rpkm was calculated by Lifescope 2.5.1
Genome_build: mm9
Supplementary_files_format_and_content: tablecount.txt.gz: tab-delimited text files include conts, RPKM values of genes for each sample

source_name: Tumour biopsy

extract_protocol:
Total ARN were extracted in Qiazol and purified using the miRNeasy minikit (Qiagen).
RNA libraries were then generated with the NEB next small library prep set for SOLID

value: DMBA/TPA-induced Tumour biopsy

tag: concentration sample (ng/nl)

tag: rin number


title: P2

source_name: Papilloma biopsy

value: DMBA/TPA-induced Papilloma 2 biopsy


title: P1

value: Biosy of DMBA/TPA-induced Papilloma 1 Biopsy


title: Δura3 control 3

description: rRNA depleted total RNA


title: mSCC-38

source_name: Tumorigenic skin cell

value: skin cells isolated from DMBA/TPA tumor


title: Stat3 ChIP1

value: Primary Cortical Oligodendrocyte Progenitor (OLP) cells


title: mSCC-20

source_name: Non tumorigeneic skin cell

value: skin cells isolated and cultured from DMBA/TPA tumor


title: CAL165

data_processing:
Reads were first filtered to remove SOLiD adaptors, SOLiD barcodes and rRNAs reads.
Alignment was performed within Lifescope 2.5.1 software RNAseq pipeline using default parameters.
Raw sequence reads were counts and rpkm was calculated by Lifescope 2.5.1
Genome_build: hg19
Supplementary_files_format_and_content: tablecount.txt.gz: tab-delimited text files include conts, RPKM values of genes

source_name: Head and neck carcinoma

extract_protocol:
Total ARN were extracted in Qiazol and purified using the miRNeasy minikit (Qiagen).
RNA libraries were generated with the NEB next small library prep set for SOLID


title: PtpA-Chip sample

source_name: Monoclonal ANTI-FLAG(R) M2 antibody ChIP in U937 Cells

tag: antibody

value: Monoclonal ANTI-FLAG(R) M2 antibody


title: Δura3 control 1

description: rRNA depleted total RNA


title: Female soma replicate 3

status: Public on Jun 06 2017

city: London

first: Michelle

last: Percharde

address: Du Cane Road

department: Imperial College London

country: United Kingdom

institute: MRC Laboratory of Medical Sciences

data_processing:
Reads were quality and adaptor trimmed using trim_galore v0.4.0, and standard parameters
Trimmed reads were aligned to mm10 plus ERCC sequences (Thermo Fisher Scientific) using tophat2 v2.0.9, using standard parameters
Bam files were used to generate count data using htseq-count v0.6.1p1, parameters: -f bam -s reverse -t exon -i gene_id -m union
Count data was analyzed in R v3.3.0, using Bioconductor v3.3, Deseq2 v.1.12.3, EdgeR v3.14.0, limma v3.28.14
Genome_build: mm10
Supplementary_files_format_and_content: ERCC-normalized-genes,log2, txt file
Supplementary_files_format_and_content: Read-depth-normalized-genes,log2, txt file

source_name: E13.5 Oct4GFP gonads

growth_protocol: Mice were maintained under standard conditions. Pregnant females after mating to Oct4GFP males were sacrificied at E13.5 and embryonic gonads isolated

extract_protocol:
RNA was isolated from sorted PGCs or soma with the Rneasy Mini kit (Qiagen), according to manufacturer's protocol, and DNAse 1 treated. 4uL 1:10000 diluted ERCC spike-in mix was added to each sample per 10000 cells
Libraries were generated using the NEBnext Ultra Directional mRNA-seq kit using 8-13ng total DNAse-1 treated RNA per sample

treatment_protocol: Oct4GFP(+) PGCs or Oct4GFP(-) soma were isolated by FACS on a BD Aria II YG

value: soma

tag: Sex

value: female


title: Testes_RNAseq_rep1

description:
RNA (polyA enriched)
Testes_RNAseq

value: Testes

value: Ovation RNASeq System V2


title: Female soma replicate 1


title: Liver_RNAseq_rep1

description:
RNA (polyA enriched)
Liver_RNAseq

value: Liver


title: Male soma replicate 3

value: male


title: Female PGC replicate 1

value: primordial germ cells


title: Female PGC replicate 3


title: Male soma replicate 2


title: Female soma replicate 2


title: Male soma replicate 1


title: Male PGC replicate 3


title: S

source_name: Healthy skin biopsy

tag: tissue

value: Total skin biopsy


title: Male PGC replicate 2


title: HAEC.ChIP.JUN.oxpapc.4h

city: Tucson

first: Casey

middle: E

last: Romanoski

state: AZ

address: 1657 E. Helen St.

department: Cellular & Molecular Medicine

institute: University of Arizona

data_processing:
demultiplexed ATAC-seq and ChIP-seq fastq data were aligned to hg19 using Bowtie2.
RNA-seq data was aligned to hg19 using STAR
Aligned sam files were made into tagDirectories in HOMER and analyzed there. Reads mapping to mitochondiral DNA were discarded for ATAC seq. ATAC and ChIP-seq peaks were identified using the findPeaks function. Motif enrichment at peaks was performed using findMotifsGenome.
Genome_build: hg19
Supplementary_files_format_and_content: peak files are in the original HOMER output format (http://homer.salk.edu/homer/), and gene expression from RNA-seq experiments is normalized using RPKM within the analyzeRepeats program in HOMER.

source_name: human aortic endothelial cell

growth_protocol: HAEC were isolated from heart transplant donor specimens, and grown in culture to 90% confluence on 6-well, 10 cm, or 15 cm plates before treatment and harvesting for ChIP-seq, RNA-seq, or ATAC-seq.

extract_protocol:
For ChIP-seq, Cells were fixed with formaldehyde, lysates were sonicated, and complexes precipitated with antibody. For RNA-seq, RNA was extracted using the the Quick-RNA Micro Prep kit from ZymoResearch and poly-A selected. For ATAC-seq, nuclei were extracted with nuclear isolation buffer and DNA was size-selected for 125-175 bp fragments on a TBE gel
For RNA-seq, cDNA was synthesized using the SuperScript III system from Invitrogen, followed by second strand synthesis, ds End Repair and UDG treatment to recover strand-specific libraries. Libraries were barcoded with BioO Nextflex adapters and amplified prior to sequencing. ChIP-seq libraries were synthesized similarly omiting the UDG treatment. ATAC-seq libraries were extracted from TBE gel, amplified, and sequenced.

treatment_protocol: Cells were treated with M-199 containing 1% FBS (control), or additionally containing either 40 µg/mL Ox-PAPC, 2 ng/mL human recombinant TNFα, or 10 ng/mL human recombinant IL-1B

tag: passage

value: Santa Cruz #sc-13032X

tag: sample type


title: HAEC.ChIP.JUN.TNFa.4h


title: HAEC.ChIP.JUN.IL1b.4h


title: HAEC.rna.siIRF1.control.4h.rep1.donor3

data_processing:
demultiplexed ATAC-seq and ChIP-seq fastq data were aligned to hg19 using Bowtie2.
RNA-seq data was aligned to hg19 using STAR
Aligned sam files were made into tagDirectories in HOMER and analyzed there. Reads mapping to mitochondrial DNA were discarded for ATAC seq. ATAC and ChIP-seq peaks were identified using the findPeaks function. Motif enrichment at peaks was performed using findMotifsGenome.
peak files are in the original HOMER output format (http://homer.salk.edu/homer/), and gene expression from RNA-seq experiments is normalized using RPKM within the analyzeRepeats program in HOMER.
Genome_build: hg19
Supplementary_files_format_and_content: peak files, normalized gene expression file

tag: cell type

value: HAEC

tag: treatment

value: siIRF1, control, 4h

tag: individual


title: HAEC.rna.siIRF1.TNFa.4h.rep2.donor3

value: siIRF1, TNFa, 4h


title: HAEC.rna.siIRF1.TNFa.4h.rep1.donor3


title: HAEC.rna.siSCR.4h.donor2

value: siSCR, 4h


title: HAEC.rna.siIRF1.control.4h.rep2.donor3


title: HAEC.rna.siSCR.4h.donor1


title: HAEC.rna.siSCR.TNFa.4h.rep2.donor3

value: siSCR, TNFa, 4h


title: HAEC.rna.siSCR.control.4h.rep2.donor3

value: siSCR, control, 4h


title: HAEC.rna.siSCR.control.4h.rep1.donor3


title: HAEC.rna.siSCR.TNFa.4h.rep1.donor3


title: HAEC.rna.siERG.TNFa.4h.rep2.donor3

value: siERG, TNFa, 4h


title: Female PGC replicate 2

tag: strain


title: HAEC.rna.siERG.TNFa.4h.rep1.donor3


title: HAEC.rna.siERG.control.4h.rep2.donor3

value: siERG, control, 4h


title: HAEC.rna.siERG.4h.donor2.rep2

value: siERG, 4h


title: HAEC.rna.siERG.control.4h.rep1.donor3


title: HAEC.rna.siCEBPD.TNFa.4h.rep1.donor3

value: siCEBPD, TNFa, 4h


title: Male PGC replicate 1


title: HAEC.rna.siERG.4h.donor2.rep1


title: HAEC.rna.mock.4h.donor2.rep2

value: mock, 4h


title: HAEC.rna.siERG.4h.donor1


title: HAEC.rna.mock.4h.donor2.rep1


title: HAEC.rna.tnfa.4h.rep1

value: tnfa, 4h


title: HAEC.ChIP.ERG.TNFa.4h

tag: chip antibody

value: Santa Cruz #sc354


title: HAEC.rna.oxpapc.4h.rep2

value: oxpapc, 4h


title: HAEC.rna.siCEBPD.TNFa.4h.rep2.donor3


title: HAEC.rna.siCEBPD.control.4h.donor3

value: siCEBPD, control, 4h


title: HAEC.rna.il1b.4h.rep2

value: il1b, 4h


title: HAEC.rna.oxpapc.4h.rep1


title: HAEC.rna.tnfa.4h.rep2


title: HAEC.rna.il1b.4h.rep1


title: HAEC.input.TNFa.4h.formaldehyde

value: TNFa, 4h, formaldehyde


title: Cardiomyocytes-D8

city: Cambridge

first: Adam

middle: Ezra

last: Cohen

address: 12 Oxford Street

department: Chemistry and Chem. Biol.

institute: Harvard University

data_processing:
Libraries were sequenced to an average depth of 568,867 paired-end reads of length 25 bp
The reads were mapped to the UCSC human transcriptome (genome build hg19) using RSEM v1.2.19 and Bowtie v1.1.1 as the read mapper
For all genes, read counts and transcript-per-million (TPM) were retrieved
Genome_build: hg19

source_name: Human induced pluripotent stem cell-derived cardiomyocytes

growth_protocol: HiPS-CM (iCell cardiomyocytes), plating and maintenance medium were purchased from Cellular Dynamics International. Cells were thawed and cultured following manufacturer instructions, and maintained at 37 °C with 5% CO2.  Cells were plated at a density of 50k/cm2, calibrated by a Trypan blue stain. Cells were plated initially in just the recessed region of the glass-bottomed dish.  After 1 hour, a very gentle rinsing removed dead and un-adhered cells and the volume of plating medium was increased to 1 mL. 48 hours after plating, the plating medium was aspirated and replaced with maintenance medium. While performing the medium exchange, extra care is required to prevent the patterned cardiomyocytes from lifting off the dish. The medium was exchanged every other day after plating.

extract_protocol:
Total RNA was extracted using a RNeasy plus Micro RNA kit (Qiagen) followed by RT-PCT to produce cDNA
Libraries were prepared using the SMART-seq2 protocol modified to use Maxima H Minus enzyme for reverse transcription (Thermo Scientific) as follows: population controls were generated by extracting total RNA using a RNeasy plus Micro RNA kit (Qiagen) according to manufacturer’s recommendations, and 1 uL of RNA in water was added to 3 uL of elution mix.  Amplified cDNA products were purified using AMPure XP SPRI beads (Beckman Coulter) and eluted in TE buffer (Teknova). Cleaned-up amplified cDNA was used for library construction using the Nextera XT DNA Sample Preparation Kit and Nextera XT Index Kit (Illumina). Libraries were then pooled and cleaned up using AMPure XP SPRI beads (Beckman Coulter Genomics). Final library quality and quantity were assessed using a DNA High Sensitivity chip (Agilent).

treatment_protocol: Cardiomyocytes were infected with CaViar constructs 10 days after plating by adding 100 μL of the lentivirus and imaged 4 days post viral infection.  The cardiomyocyte medium was replaced with fresh maintenance medium 24 hours after adding the virus. Details of the CaViar construct: The CaViar construct was cloned into the pLenti-CMV-puro plasmid (addgene #17448).  This backbone is a third generation lentiviral plamsid with puromycin resistance that drives transgene expression under a CMV promoter. The plasmid was cut with PmeI and SalI and purified by gel elution.   The CaViar construct is a fusion of v5 epitope tag, QuasAr2, the Kir2.1 trafficking sequence (TS), and GCaMP6f.


title: Cardiomyocytes-D9

value: Human induced pluripotent stem cell-derived cardiomyocytes


title: Cardiomyocytes-D7


title: HAEC.rna.control.4h.rep1

value: HAEC

value: control, 4h


title: HAEC.input.oxpapc.4h.formaldehyde

value: oxpapc, 4h, formaldehyde


title: Cardiomyocytes-D3


title: Cardiomyocytes-D10


title: Cardiomyocytes-D6


title: HAEC.rna.control.4h.rep2


title: Cardiomyocytes-D4


title: HAEC.input.TNFa.4h.DSG

value: TNFa, 4h, DSG


title: Cardiomyocytes-D5


title: HAEC.input.IL1b.4h.formaldehyde

value: IL1b, 4h, formaldehyde


title: Cardiomyocytes-B10


title: Cardiomyocytes-D1


title: Cardiomyocytes-D2


title: Cardiomyocytes-B9


title: Cardiomyocytes-B8


title: Zs_neg_48hr_rep1

first: Zachary

last: Carico

address: 207 Research Drive

department: Immunology

data_processing:
Basecalls and demultiplexing performed using Illumina MiSeq on-device software.
Raw sequences were aligned to reference library of strain 129 V-alpha, V-delta, and J-alpha gene segments, as cataloged at www.imgt.org, using MiXCR version 1.8, align routine with parameter '-p rna-seq'.
Individual TCR-alpha clones were identified from aligned sequences using MiXCR version 1.8, assemble routine with parameter ‘-OassemblingFeatures=[CDR2+FR3+CDR3]’.
Individual clones identified by the MiXCR assemble routine were written in human-readable format using MiXCR exportClones routine.
Trav16 and Trav16d alignments were performed manually within Microsoft Excel.
Genome_build: Custom strain 129 V-alpha, V-delta, and J-alpha reference library.
Supplementary_files_format_and_content: .txt files with sequences, gene segment identities, and counts of unique Tcra clones.

source_name: DP thymocytes

growth_protocol: 4-week-old mice were injected i.p. with 1 mg tamoxifen 48 hours prior to sacrifice.

extract_protocol:
Total RNA was extracted from sorted DP thymocytes using Trizol (Life Technologies).
Adapters were ligated to the 5' ends of total RNA using a template-switch RACE protocol. Tcra transcripts were amplified, and Illumina adapter sequences added, using a sense primer that annealed to the 5' RACE adapter and an antisense primer that annealed to Trac.

tag: genotype/variation

value: Tcrd-CreER x Rosa26-fl-STOP-fl-ZsGreen

value: thymus

value: DP thymocytes


title: Cardiomyocytes-B11


title: Zs_neg_48hr_rep2


title: HAEC.input.IL1b.4h.DSG

value: IL1b, 4h, DSG


title: Zs_neg_24hr_rep1

growth_protocol: 4-week-old mice were injected i.p. with 1 mg tamoxifen 24 hours prior to sacrifice.


title: HAEC.input.control.4h.formaldehyde

value: control, 4h, formaldehyde


title: Cardiomyocytes-B6


title: Cardiomyocytes-B7


title: Zs_neg_12hr_rep1

growth_protocol: 4-week-old mice were injected i.p. with 1 mg tamoxifen 12 hours prior to sacrifice.


title: HAEC.input.control.4h.DSG

value: control, 4h, DSG


title: Cardiomyocytes-B5


title: Cardiomyocytes-B4


title: Cardiomyocytes-B2


title: Cardiomyocytes-B3


title: Zs_pos_72hr_rep2

growth_protocol: 4-week-old mice were injected i.p. with 1 mg tamoxifen 72 hours prior to sacrifice.


title: HAEC.ChIP.p65.TNFa.4h.rep1

value: p65, Santa Cruz #sc-372X

value: TNFa, 4h


title: HAEC.ChIP.p65.TNFa.4h.rep2


title: Zs_pos_24hr_rep2


title: HAEC.ChIP.p65.IL1b.4h.rep1

value: IL1b, 4h


title: HAEC.ChIP.p65.control.4h.rep2


title: Zs_pos_48hr_rep1


title: Cardiomyocytes-B1


title: HAEC.ChIP.p65.IL1b.4h.rep2


title: Zs_pos_72hr_rep1


title: Zs_pos_48hr_rep2


title: Zs_pos_24hr_rep1


title: HAEC.ChIP.NRF2.oxpapc.4h.rep2

value: NRF2, Santa Cruz #sc-1694X

value: oxpapc, 4h


title: HAEC.ChIP.p65.control.4h.rep1


title: Zs_pos_12hr_rep1


title: HAEC.ChIP.JUNB.control.4h

value: JUNB, Santa Cruz #sc-73


title: HAEC.ChIP.NRF2.oxpapc.4h.rep1

value: HAEC


title: INT_Tcra_rep3

growth_protocol: Thymocytes were harvested from 4-week-old mice.


title: INT_Tcra_rep2


title: genomic_input_OregonR

city: Chapel Hill

first: Spencer

middle: L

last: Nystrom

state: NC

address: 250 Bell Tower Drive

department: Curriculum in Genetics and Molecular Biology

institute: UNC-Chapel Hill

description: WT-Input-DNA

data_processing:
RNAseq - Sequenced reads were mapped to the dm3 whole genome using Tophat2 v2.1.0., supplementing a dm3 version transcript annotation file and using library-type fr-firststrand
RNAseq – The resulting bam file was indexed and sorted using samtools v1.2
RNAseq – htseq-count v0.6.1 was used to count reads mapping to genes, using the parameters -s reverse -a 10 -t exon -i gene_name -f bam -r pos
RNAseq – FPKMs were calculated from the resulting count files using EdgeR
FAIREseq – Sequenced reads were mapped ot the dm3 whole genome using bowtie2 v2.2.8, using seed 123
FAIREseq – the resulting bam file was indexed and sorted using samtools v1.3.1, and all reads with a mapq score less than 5 were removed setting -bq 5
FAIREseq – the resulting bam file was indexed and sorted using samtools v1.3.1
FAIREseq – PCR duplicates were marked in the resulting sorted, indexed sam file using Picard v2.2.4 MarkDuplicates
FAIREseq – PCR duplicates were removed with samtools v1.3.1 by removing all reads marked with the PCR duplicates bit flag (samtools view -bF 0x400)
FAIREseq – The resulting bam file was indexed and further filtered to include only reads mapping to chr2L, chr2R, chr3L, chr3R, chr4, and chrX (no Y, U, or heterochromatic regions)
FAIREseq – the resulting bam file was indexed and converted to a bed file with bedtools v2.25.0
FAIREseq – bigwigs normalized to reads per genomic content (RPGC) were generated from the resulting bedfile using Deeptools v2.24.0 bamCoverage setting: --normalizeTo1x 121400000 –binSize 10 -e 125 –outFileFormat bedgraph
FAIREseq – bigwigs were Z-normalized using standard deviations and means for each chromosome arm
FAIREseq – peak calls were generated by pooling the bedfiles for each replicate which were then input to MACS2 (v 2015-06-03) using -g dm, --nomodel, --extsize 125, --seed 123
ChIPseq – Sequenced reads were mapped ot the dm3 whole genome using bowtie2 v2.2.8, using seed 123
ChIPseq – the resulting bam file was indexed and sorted using samtools v1.3.1, and all reads with a mapq score less than 5 were removed setting -bq 5
ChIPseq – the resulting bam file was indexed and sorted using samtools v1.3.1
ChIPseq – PCR duplicates were marked in the resulting sorted, indexed sam file using Picard v2.2.4 MarkDuplicates
ChIPseq – PCR duplicates were removed with samtools v1.3.1 by removing all reads marked with the PCR duplicates bit flag (samtools view -bF 0x400)
ChIPseq – The resulting bam file was indexed and further filtered to include only reads mapping to chr2L, chr2R, chr3L, chr3R, chr4, and chrX (no Y, U, or heterochromatic regions)
ChIPseq – the resulting bam file was indexed and converted to a bed file with bedtools v2.25.0
ChIPseq – bigwigs normalized to reads per genomic content (RPGC) were generated from the resulting bedfile using Deeptools v2.24.0 bamCoverage setting: --normalizeTo1x 121400000 –binSize 10 -e 125 –outFileFormat bedgraph
ChIPseq – bigwigs were Z-normalized using standard deviations and means for each chromosome arm
ChIPseq – peak calls were generated by pooling the bedfiles for each replicate which were then input to MACS2 (v 2015-06-03) using -g dm, --nomodel, --extsize 125, --seed 123
MACS2 peak calls were all normalized to Control_GenomicDNA_UMR_sorted_PCRDupsRemoved.bed
Genome_build: dm3
Supplementary_files_format_and_content: RNAseq – a single matrix table in the form of a xls file contains FPKMs for genes at all the timepoints
Supplementary_files_format_and_content: FAIREseq – Z-normalized bigwig files generated from the pooled datasets used to generate peakCalls
Supplementary_files_format_and_content: FAIREseq – txt files containing output from MACS2 peak calls on pooled datasets
Supplementary_files_format_and_content: ChIPseq – txt files containing output from MACS2 peak calls on pooled datasets

organism: Drosophila melanogaster

source_name: adult female virgins

extract_protocol: Rubicon Thruplex DNA-seq kit

value: whole adult

tag: developmental stage

value: adult

value: OregonR

value: Wild-type

tag: biological replicate number


title: INT_Tcra_rep1


title: HAEC.ChIP.JUN.control.4h.rep2

value: JUN, Santa Cruz #sc-13032X

value: control, 4h


title: HAEC.ChIP.JUN.control.4h.rep1


title: HAEC.ChIP.IRF1.TNFa.4h

value: IRF1, Santa Cruz #sc-497x


title: HY_Tcra_spl_rep2

source_name: naïve T cells

value: HYa

value: spleen

value: naive T cells


title: HAEC.ChIP.IRF1.IL1b.4h


title: HAEC.ChIP.H3K4me2.control.4h.rep2

value: H3K4me2, EMD Millipore #07-030


title: HY_Tcra_spl_rep1


title: Zs_pos_12hr_rep2

value: Tcrd-CreER x Rosa26-fl-STOP-fl-ZsGreen

value: thymus

value: DP thymocytes


title: HAEC.ChIP.H3K4me2.control.4h.rep1


title: HAEC.ChIP.H3K27ac.oxpapc.4h.rep1

value: H3K27ac, Active Motif #39135

value: oxpapc, 4h


title: HAEC.ChIP.H3K27ac.IL1b.4h.rep1

value: HAEC

value: IL1b, 4h


title: HAEC.ChIP.H3K27ac.TNFa.4h.rep2

value: TNFa, 4h


title: HAEC.ChIP.H3K27ac.TNFa.4h.rep1


title: WT_Tcra_rep2

tag: strain/variation

value: WT


title: HY_Tcra_thy_rep1


title: HAEC.ChIP.ERG.control.4h.rep2

value: ERG, Santa Cruz #sc354


title: WT_Tcra_rep1


title: HAEC.ChIP.H3K27ac.control.4h.rep1


title: HAEC.ChIP.H3K27ac.control.4h.rep2


title: WT_Tcra_rep3


title: HAEC.ChIP.H3K27ac.IL1b.4h.rep2


title: HY_Tcra_thy_rep2


title: HAEC.atac.tnfa.4h.rep2

value: tnfa, 4h


title: HAEC.ChIP.CEBPD.IL1b.4h

value: CEBPD, Santa Cruz #sc-636X


title: HAEC.ChIP.ERG.control.4h.rep1

value: control, 4h


title: HAEC.atac.oxpapc.4h.rep2


title: HAEC.atac.tnfa.4h.rep1


title: HAEC.atac.il1b.4h.rep2

value: il1b, 4h


title: HAEC.atac.control.4h.rep2


title: HAEC.atac.il1b.4h.rep1


title: HY_Tcra_thy_rep3

value: HYa


title: HY_Tcra_thy_rep4


title: HAEC.ChIP.H3K27ac.oxpapc.4h.rep2


title: HAEC.atac.control.4h.rep1


title: sickle R. solani inoculated treated 7 dpi rep 1

city: Floreat

first: Jonathan

middle: P

last: Anderson

state: Western Australia

address: 147 Underwood Avenue

country: Australia

institute: CSIRO

data_processing:
Image analysis was performed in real time by the HiSeq Control Software (HCS) v2.2.68 and Real Time Analysis (RTA) v1.18.66.3. Then the Illumina  bcl2fastq 2.17.1.14 pipeline was used to generate the sequence data
RNA-seq paired-end reads were trimmed for Illumina adapter sequences with reads trimmed to less than 25 bp discarded using Cutadapt (Martin, 2011) (parameters: --format fastq --overlap 10 --times 3 --minimum-length 25).
Trimmed RNA-seq reads were then mapped to M. truncatula genome version 4.0 (Tang et al., 2014) using Tophat2 (parameters: --b2-very-sensitive -r 80 --mate-std-dev 40 -i 20 -I 5000 -g 20 --report-secondary-alignments -m 0 --min-coverage-intron 20 --microexon-search --library-type fr-firststrand) (Trapnell et al., 2009; Trapnell et al., 2012). BAM files generated from Tophat2 output were sorted using SAMtools version 0.1.19 (Li et al., 2009).
Htseq-count (Anders et al., 2015) was used to count aligned reads per gene and the resulting count matrix was normalized and analyzed for differential expression using the EdgeR package (Robinson et al., 2010) in R version 3.2.2 using a 0.05 FDR cut-off to determine differentially expressed genes.
Genome_build: Medtr version 4.0
Supplementary_files_format_and_content: Tab delimited matrix showing average log2fold change for inoculated samples over mock samples and the associated FDR from incorporation of all three replicates for each genotype/time point combination.

organism: Medicago truncatula

source_name: Seedling root tissue

growth_protocol: M. truncatula lines A17 and skl were grown according to (Anderson et al., 2010, Plant Physiol. 154:861-873). Surface sterilised M. truncatula seeds were germinated on moist filter paper in the dark at 4 °C for two days, then one day at 24 °C.

extract_protocol:
Frozen root tissue was ground in the presence of liquid nitrogen and total RNA extracted using Trizol reagent (Sigma) according to manufacturer’s instructions.
Library preparation and sequencing was conducted by the Australian Genome Research Facility or Beijing Genome Institute using the TruSeq Stranded Total RNA Library Kit (Illumina)

treatment_protocol: R. solani AG8 (isolate WAC10335) was prepared according to (Lichtenzveig et al., 2006). Seedlings were added to pots half filled with vermiculite also containing 5 ml of 2.2mg/ml dry weight equivalent suspension of homogenised fungal mycelium. The seedlings were covered with fine vermiculite and transferred to growth rooms at a constant temperature of 24 °C with 16/8 light/dark photoperiod at a light intensity of 200 mmol m-2 s-1. Mock treated plants were treated the same with the addition of 5 ml sterile water rather than the 5 ml of R. solani suspension. Inoculated plants were harvested at two and seven days after planting into pre-inoculated pots with corresponding mock samples taken at the same time. Root tissue was collected and immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction. All experiments included three biological replicates.

tag: plant genotype

value: sickle mutant (ethylene insensitive, point mutation in A17)

tag: days after infection/mock treatment

value: 7 days

tag: disease phenotype

value: Highly susceptible to R. solani AG8


title: sickle R. solani inoculated treated 7 dpi rep 2


title: sickle Mock treated 7 dpi rep 2


title: sickle Mock treated 7 dpi rep 3


title: sickle Mock treated 7 dpi rep 1


title: sickle R. solani inoculated treated 2 dpi rep 1

value: 2 days


title: sickle R. solani inoculated treated 2 dpi rep 2


title: sickle R. solani inoculated treated 2 dpi rep 3


title: A17 R. solani inoculated treated 7 dpi rep 3

value: A17 (wild type)

value: moderately resistant to R. solani AG8


title: sickle Mock treated 2 dpi rep 1


title: HAEC.atac.oxpapc.4h.rep1

value: oxpapc, 4h


title: A17 Mock treated 7 dpi rep 1


title: A17 R. solani inoculated treated 2 dpi rep 1


title: A17 Mock treated 7 dpi rep 2


title: A17 R. solani inoculated treated 7 dpi rep 2


title: A17 R. solani inoculated treated 2 dpi rep 2


title: A17 R. solani inoculated treated 2 dpi rep 3


title: sickle Mock treated 2 dpi rep 3


title: A17 Mock treated 2 dpi rep 2


title: A17 Mock treated 2 dpi rep 1


title: A17 Mock treated 7 dpi rep 3


title: A17 Mock treated 2 dpi rep 3


title: WT-44hAPF-RNA-Rep1

description:
WT-44hAPF-RNA-Rep1
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: GGCTAC

source_name: 44hAPF wings

extract_protocol:
A minimum of 40 wings were dissected from staged female flies in 1x PBS. RNA was isolated using Trizol.
Rubicon Thruplex DNA-seq kit

value: wing

tag: genotype

value: Wild-type


title: WT-44hAPF-RNA-Rep2

description:
WT-44hAPF-RNA-Rep2
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: CTTGTA


title: WT-24hAPF-RNA-Rep1

description:
WT-24hAPF-RNA-Rep1
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: CAGATC

source_name: 24hAPF wings


title: WT-24hAPF-RNA-Rep2

description:
WT-24hAPF-RNA-Rep2
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: ACTTGA


title: WT-3LW-RNA-Rep2

description:
WT-3LW-RNA-Rep2
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: CGATGT

source_name: 3LW wing imaginal discs


title: WT-44hAPF-FAIRE-Rep3

description:
WT-44hAPF-FAIRE-Rep3
WT_44hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_44hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: GCCAAT

extract_protocol:
A minimum of 40 wings were dissected from staged female flies in 1x PBS and transferred to ice for subsequent processing. 6 rounds of bead beating (1 minute beating, 2 minutes resting) were performed on dissected tissue. Lysate was sonicated with a Branson sonifier to a fragment size range of 500 bp – 2kb. Soluble chromatin fraction was isolated by centrifugation at 15,000 RCF x 5’ at 4C. Soluble chromatin fraction was phenol-chloroform extracted. Isolated FAIRE-enriched DNA was used for library prep.
Rubicon Thruplex DNA-seq kit


title: WT-44hAPF-FAIRE-Rep2

description:
WT-44hAPF-FAIRE-Rep2
WT_44hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_44hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: ACAGTG


title: sickle Mock treated 2 dpi rep 2


title: A17 R. solani inoculated treated 7 dpi rep 1


title: WT-44hAPF-FAIRE-Rep1

description:
WT-44hAPF-FAIRE-Rep1
WT_44hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_44hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: CTTGTA


title: WT-24hAPF-FAIRE-Rep2

description:
WT-24hAPF-FAIRE-Rep2
WT_24hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_24hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: ATCACG


title: WT-24hAPF-FAIRE-Rep1

description:
WT-24hAPF-FAIRE-Rep1
WT_24hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_24hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: TAGCTT


title: WT-24hAPF-FAIRE-Rep3

description:
WT-24hAPF-FAIRE-Rep3
WT_24hAPF_RepPooled_PCRDupsRemoved_peaks.txt
WT_24hAPF_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: CGATGT


title: WT-3LW-FAIRE-Rep3

description:
WT-3LW-FAIRE-Rep3
WT_3LW-Rep123-Pooled_peaks.txt
WT_3LW_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: GATCAG


title: S2 T0 RNA-seq rep2

city: Boston

first: Michael

last: Tolstorukov

address: 185 Cambridge Street

department: Molecular Biology

institute: Massachusetts General Hospital

description: same raw and processed data files as in GSM2083104

data_processing:
MNase-seq, ATAC-seq and ChIP-seq:  The sequenced paired-end reads were mapped to dm3 genome using Bowtie aligner v. 0.12.939. Only uniquely mapped reads with no more than two mismatches were retained.  The reads with the insert sizes less than 50 bp or larger than 500 bp were filtered out. Genomic positions with the numbers of mapped tags above the significance threshold of z-score = 7 were identified as anomalous, and the tags mapped to such positions were discarded. Read frequencies were computed in 300-bp non-overlapping bins in the case of fly data and in 500-bp bins in the case of human data for each titration point independently. The read frequencies were normalized by the corresponding library sizes to represent values per one million of mapped reads. To facilitate the comparison of the results between different genomes, the frequencies were additionally scaled by the factors representing ratios between the corresponding genome size and 100 Mb, similarly to the approach described in Kasinathan et al. (Nat. Methods 2014).
RNA-seq:  Tags were aligned using Tophat software package with default parameters. RNA-Seq tag frequencies were normalized for GC-content using bioconductor package EDASeq and then the expression estimates for each gene were obtained using bioconductor package DESeq.
Genome_build: dm3
Supplementary_files_format_and_content: bedGraph files representing coverage profiles normalized to library sizes

source_name: UPR-induced S2 cells, T0 RNA-seq

extract_protocol: [RNA-seq] 10^7 Drosophila S2 cells were harvested on ice, washed in cold PBS and their RNA was extracted using the RNeasy Kit (Qiagen) with on column DNAse digestion according to the manufacturer’s protocol. Ribosomal RNA was depleted from the total RNA (5ug) by using the RiboZero gold magnetic kit (Epicentre/Illumina) according to manufacturer’s instructions. cDNA was generated with the TruSeq non–stranded kit (Illumina) according to manufacturer’s instructions

value: S2 cell

tag: time

value: T0 (0 hours)


title: WT-3LW-FAIRE-Rep1

description:
WT-3LW-FAIRE-Rep1
WT_3LW-Rep123-Pooled_peaks.txt
WT_3LW_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: GCGAT

tag: tissue


title: Serum/LIF -GMEM_129_MethylC-seq

first: Charles

last: Bradshaw

address: Tennis Court Road

department: Gurdon Institute

institute: University of Cambridge

description: MethylC-seq

data_processing:
Reads were filtered to remove all reads with a quality score of < Q20.
Mapping was performed with Tophat 2.0.6
RPKMs were generated from the ucsc mm10 gtf
Bisulfite mapping was performed with Bismark 0.7.12
CpG calls were extracted with Bismark methylation extractor
Genome_build: mm10
Supplementary_files_format_and_content: text file with RPKM values or Bismark output

source_name: Serum/LIF -GMEM_129

growth_protocol: Cell sample specific- culture parameters included in title

extract_protocol:
Total RNA was extracted using RNAeasy mini kit (Qiagen) with on-column Dnase step, and 1μg was used for polyA RNA library construction.
Libraries were prepared according to Illumina TruSeq v2.0 protocols

tag: passages


title: S2 T0 RNA-seq rep1

description: same raw and processed data files as in GSM2083103


title: WT-3LW-FAIRE-Rep2

description:
WT-3LW-FAIRE-Rep2
WT_3LW-Rep123-Pooled_peaks.txt
WT_3LW_FAIRE_merge_Rep123_bs10_e125_1x_znorm.bw
Barcode: ACAGG


title: S2 T4 Pol-II input rep1

data_processing:
MNase-seq, ATAC-seq and ChIP-seq:  The sequenced paired-end reads were mapped to dm3 genome using Bowtie aligner v. 0.12.939. Only uniquely mapped reads with no more than two mismatches were retained.  The reads with the insert sizes less than 50 bp or larger than 500 bp were filtered out. Genomic positions with the numbers of mapped tags above the significance threshold of z-score = 7 were identified as anomalous, and the tags mapped to such positions were discarded. Read frequencies were computed in 300-bp non-overlapping bins in the case of fly data and in 500-bp bins in the case of human data for each titration point independently. The read frequencies were normalized by the corresponding library sizes to represent values per one million of mapped reads. To facilitate the comparison of the results between different genomes, the frequencies were additionally scaled by the factors representing ratios between the corresponding genome size and 100 Mb.
RNA-seq:  Tags were aligned using Tophat software package with default parameters. RNA-Seq tag frequencies were normalized for GC-content using bioconductor package EDASeq and then the expression estimates for each gene were obtained using bioconductor package DESeq.
Genome_build: dm3
Supplementary_files_format_and_content: bedGraph files representing coverage profiles normalized to library sizes

source_name: UPR-induced S2 cells, T4 Pol-II input

extract_protocol: [H3K27ac and RNA Polymerase II protocol] 3-6 x 10^7 crosslinked cells from each treatment group were resuspended in sonication buffer (0.5% SDS, 20mM Tris pH8.0, 0.5 mM EGTA, 2mM EDTA and protease inhibitor tablets (Roche)) in a ratio of 100ul cold sonication buffer to 1x10^7 cells. Cells were lysed on ice for 10min. Cell lysates were sonicated in a Qsonica at 4C. After a hard spin for 10 min at 4C, the supernatants corresponding to 2.5 million cells were removed and either flash frozen and kept at -80 (as input) or diluted into 1ml of ChIP buffer (0.5% Triton X-100, 2mM EDTA, 20mM TRIS pH8.0, 150mM NaCl, 10% glycerol) to be used for chromatin IP after a hard spin for 10min at 4C. The IP solution was incubated with selected IP antibody at 4C overnight tumbling. The next day proteinA dynabeads (Life Technologies) were added and after a 2hr incubation while tumbling at 4C, the IP samples were washed once with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH8, 150mM NaCl), three times with high salt wash buffer (same as previous buffer but with 500mM NaCl), and once with LiCl wash buffer (0.25M LiCl, 1% NP-40, 1% NADeoxycholate, 1mM EDTA, 10mM Tris pH 8). The beads were rinsed with TE buffer and the proteins were eluted with 500ul elution buffer (1%SDS, 0.1M NaHCO3) for 30 min at room temperature. At this point the input samples were also taken up in elution buffer.

value: T4 (4 hours)


title: S2 T1 RNA-seq rep2

source_name: UPR-induced S2 cells, T1 RNA-seq

value: T1 (1 hour)


title: S2 T4 RNA-seq rep1

source_name: UPR-induced S2 cells, T4 RNA-seq


title: S2 T1 RNA-seq rep1


title: S2 T4 RNA-seq rep2


title: S2 T1 Pol-II input rep1

source_name: UPR-induced S2 cells, T1 Pol-II input


title: S2 T4 Pol-II ChIP rep2

source_name: UPR-induced S2 cells, T4 Pol-II ChIP

value: Pol-II (abcam ab5131)


title: S2 T0 Pol-II input rep2

source_name: UPR-induced S2 cells, T0 Pol-II input

value: T0 (0 hours, control)


title: Serum/LIF – KO DMEM_129_MethylC-seq

description: MethylC-seq

source_name: Serum/LIF – KO DMEM_129


title: S2 T0 Pol-II input rep1


title: S2 T4 Pol-II ChIP rep1


title: S2 T1 Pol-II ChIP rep1

source_name: UPR-induced S2 cells, T1 Pol-II ChIP


title: S2 T0 Pol-II ChIP rep2

source_name: UPR-induced S2 cells, T0 Pol-II ChIP


title: S2 T0 Pol-II ChIP rep1


title: WT-3LW-RNA-Rep1

description:
WT-3LW-RNA-Rep1
geneFPKMs_L3_24hr_44hr.xlsx
Barcode: ATCACG


title: S2 T4 H3K27ac input rep2

source_name: UPR-induced S2 cells, T4 H3K27ac input


title: Ch/LIF – N2B27_129_MethylC-seq

description: MethylC-seq

source_name: Ch/LIF – N2B27_129


title: Pd/LIF – N2B27_129_MethylC-seq

description: MethylC-seq

source_name: Pd/LIF – N2B27_129


title: Serum/LIF – Feeders - GMEM_129_MethylC-seq

description: MethylC-seq

source_name: Serum/LIF – Feeders - GMEM_129


title: 2i/LIF – N2B27_129_MethylC-seq

description: MethylC-seq


title: 2i/LIF – GMEM/10% serum_129_MethylC-seq

description: MethylC-seq

source_name: 2i/LIF – GMEM/10% serum_129


title: S2 T4 H3K27ac input rep1


title: S2 T4 Pol-II input rep2


title: 2i/LIF – GMEM/10% KSR_129_MethylC-seq

description: MethylC-seq

source_name: 2i/LIF – GMEM/10% KSR_129


title: 2i – N2B27_129_MethylC-seq

description: MethylC-seq


title: S2 T0 H3K27ac input rep2

source_name: UPR-induced S2 cells, T0 H3K27ac input


title: Serum/LIF – GMEM_BL6_Rep1

description: RNA-seq

source_name: Serum/LIF – GMEM_BL6_Rep1


title: S2 T4 H3K27ac ChIP rep2

source_name: UPR-induced S2 cells, T4 H3K27ac ChIP

value: H3K27ac (active motif 39136)


title: Serum/LIF – GMEM_129_Rep2

description: RNA-seq

source_name: Serum/LIF – GMEM_129_Rep2


title: Serum/LIF – KO DMEM_BL6_Rep1

description: RNA-seq

source_name: Serum/LIF – KO DMEM_BL6_Rep1


title: Serum/LIF – GMEM_129_Rep1

description: RNA-seq

source_name: Serum/LIF – GMEM_129_Rep1


title: S2 T1 H3K27ac ChIP rep1

source_name: UPR-induced S2 cells, T1 H3K27ac ChIP


title: S2 T1 H3K27ac ChIP rep2


title: S2 T4 H3K27ac ChIP rep1


title: S2 T0 H3K27ac ChIP rep2

source_name: UPR-induced S2 cells, T0 H3K27ac ChIP


title: S2 T0 H3K27ac input rep1


title: PIK2

city: Seattle

first: Slobodan

last: Beronja

state: WA

address: 1100 Fairview Ave. N

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

data_processing:
Standard Illumina outupt fastq
tophat map to mm9
Cufflinks determine FPKM
Genome_build: mm9
Supplementary_files_format_and_content: txt. Contain gene level FPKM output from Cufflinks

extract_protocol:
FACS sorted a6 integrin high basal cells followed by Qiaamp RNEAsy Kit protocol
RNA libraries were prepared for sequencing using standard Illumina protocols

value: a6 integrin high basal cells


title: Serum/LIF – KO DMEM_BL6_Rep2

description: RNA-seq

source_name: Serum/LIF – KO DMEM_BL6_Rep2


title: PIK1


title: Serum/LIF – KO DMEM_129_Rep1

description: RNA-seq

source_name: Serum/LIF – KO DMEM_129_Rep1


title: WT2

value: WT


title: S2 T1 H3K27ac input rep2

source_name: UPR-induced S2 cells, T1 H3K27ac input


title: S2 T4 ATAC-seq rep2

source_name: UPR-induced S2 cells, T4 ATAC-seq

extract_protocol: [ATAC-seq] 5x10^4 S2 cells from each time point were collected and washed in cold PBS. ATAC-Seq libraries were prepared as described previously (Buenrostro et al. Nat. Methods 2013; Buenrostro et al. Curr Protoc Mol Biol. 2015). Briefly, cells were resuspended in cold lysis buffer and centrifuged to collect nuclei. Transposition reaction was performed for 30 minutes at 37°C using Tn5 transposase from Nextera. DNA fragments were purified using a Minelute kit (Qiagen). DNA fragments were amplified using Nextera barcoded primers as described. Amplified library was purified with SPRI beads. Paired End 50 sequencing was done on an Illumina HiSeq2000 according to manufacturer’s instructions.


title: WT1


title: Serum/LIF – Feeders - GMEM_BL6_Rep1

description: RNA-seq

source_name: Serum/LIF – Feeders - GMEM_BL6_Rep1


title: S2 T4 ATAC-seq rep1


title: CF_Tam-rep3

city: Carlsbad

first: Sagar

middle: S

last: Damle

state: CA

address: 2855 Gazelle Ct

institute: Ionis Pharmaceuticals

description: inducible knockout (Tamoxifen)

data_processing:
Sequenced reads were mapped using rnaSTAR to the mouse reference genome
Differentially expressed genes were identified using cufflinks with default parameters
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: tab-delimited cufflinks transcript abundance files including FPKM values for each sample

growth_protocol: Mouse hepatocytes were obtained by liver perfusion in Hank's Balanced Salt Solution followed by collagenase

extract_protocol:
Total RNA was isolated from hepatocytes using miRNease mini kit (Qiagen)
RNAseq libraries were constructed using illumina Stranded total RNA library kit

value: induced RNASEH1 exon 4/5 deletion

value: liver

value: hepatocyte


title: Serum/LIF – Feeders - GMEM_BL6_Rep2

description: RNA-seq

source_name: Serum/LIF – Feeders - GMEM_BL6_Rep2


title: Serum/LIF -GMEM_BL6_MethylC-seq

description: MethylC-seq

source_name: Serum/LIF -GMEM_BL6


title: Serum/LIF – GMEM_BL6_Rep2

description: RNA-seq

source_name: Serum/LIF – GMEM_BL6_Rep2


title: S2 T1 H3K27ac input rep1


title: Serum/LIF – KO DMEM_129_Rep2

description: RNA-seq

source_name: Serum/LIF – KO DMEM_129_Rep2


title: Serum/LIF – Feeders - GMEM_129_Rep2

description: RNA-seq

source_name: Serum/LIF – Feeders - GMEM_129_Rep2


title: PIK3


title: Serum/LIF – Feeders - GMEM_129_Rep1

description: RNA-seq

source_name: Serum/LIF – Feeders - GMEM_129_Rep1


title: Pd/LIF – N2B27_BL6_Rep2

description: RNA-seq

source_name: Pd/LIF – N2B27_BL6_Rep2


title: CF_Tam-rep2

description: inducible knockout (Tamoxifen)


title: Serum/LIF – Feeders - GMEM_BL6_MethylC-seq

description: MethylC-seq

source_name: Serum/LIF – Feeders - GMEM_BL6


title: S2 T0 H3K27ac ChIP rep1


title: S2 T0 ATAC-seq rep2

source_name: UPR-induced S2 cells, T0 ATAC-seq


title: S2 T1 ATAC-seq rep1

source_name: UPR-induced S2 cells, T1 ATAC-seq


title: Serum/LIF – KO DMEM_BL6_MethylC-seq

description: MethylC-seq

source_name: Serum/LIF – KO DMEM_BL6


title: WF_Tam-rep3

description: inducible wild type (Tamoxifen)

value: RNASEH1 WT


title: CF_Tam-rep1

description: inducible knockout (Tamoxifen)


title: WT3


title: WF_Tam-rep2

description: inducible wild type (Tamoxifen)


title: Ch/LIF – N2B27_BL6_MethylC-seq

description: MethylC-seq

source_name: Ch/LIF – N2B27_BL6


title: S2 T0 ATAC-seq rep1


title: WF_Tam-rep1

description: inducible wild type (Tamoxifen)


title: Pd/LIF – N2B27_BL6_Rep1

description: RNA-seq

source_name: Pd/LIF – N2B27_BL6_Rep1


title: Pd/LIF – N2B27_129_Rep1

description: RNA-seq

source_name: Pd/LIF – N2B27_129_Rep1


title: Pd/LIF – N2B27_129_Rep2

description: RNA-seq

source_name: Pd/LIF – N2B27_129_Rep2


title: S2 T4 rep1 traditional occupancy

source_name: UPR-induced S2 cells, T4, traditional occupancy

extract_protocol: [MNase-seq] MNase titration was performed as described previously (Mieczkowski, Cook, Bowman et al. Nat. Communications 2016). For h-MACC, after addition of EDTA/EGTA and SDS half of the digests were kept at 4C as input, the other half were adjusted to ChIP buffer conditions [10mM TRIS ph8.0, 100mM NaCl, 1mM EDTA, 0.1% sodiumdeoxycholate, 0.5% sarkosyl, 1% triton x-100 and complete protease inhibitors (Roche)] with 1ml of ChIP buffer. After tumbling for 10min at 4C, the digests were spun at high speed for 10min at 4C and the supernatant was incubated with anti H3 antibodies (abcam ab1791) as described (Bowman et al. BMC Genomics 2013).

tag: mnase concentration

value: 1.5U, 6.25U, 25U and 100U MNase titrations pooled before sequencing


title: CF-rep3

description: inducible knockout

source_name: CF

tag: cell type


title: 2i/LIF – N2B27_BL6_MethylC-seq

description: MethylC-seq

source_name: 2i/LIF – N2B27_BL6


title: Ch/LIF – N2B27_BL6_Rep2

description: RNA-seq

source_name: Ch/LIF – N2B27_BL6_Rep2


title: CF-rep2

description: inducible knockout


title: 2i/LIF – GMEM/10% serum_BL6_MethylC-seq

description: MethylC-seq

source_name: 2i/LIF – GMEM/10% serum_BL6


title: S2 T1 ATAC-seq rep2


title: 2i/LIF – GMEM/10% KSR_BL6_MethylC-seq

description: MethylC-seq

source_name: 2i/LIF – GMEM/10% KSR_BL6


title: Ch/LIF – N2B27_BL6_Rep1

description: RNA-seq

source_name: Ch/LIF – N2B27_BL6_Rep1


title: S2 T1 rep2 traditional occupancy

source_name: UPR-induced S2 cells, T1, traditional occupancy


title: Pd/LIF – N2B27_BL6_MethylC-seq

description: MethylC-seq

source_name: Pd/LIF – N2B27_BL6


title: Ch/LIF – N2B27_129_Rep2

description: RNA-seq

source_name: Ch/LIF – N2B27_129_Rep2


title: S2 T0 rep2 traditional occupancy

source_name: UPR-induced S2 cells, T0, traditional occupancy


title: Ch/LIF – N2B27_129_Rep1

description: RNA-seq

source_name: Ch/LIF – N2B27_129_Rep1


title: 2i – N2B27_BL6_MethylC-seq

description: MethylC-seq

source_name: 2i – N2B27_BL6


title: S2 T0 rep1 traditional occupancy


title: FC_6 wk-rep3

description: constitutive knockout (6 wk)

source_name: FC_6 wk

value: constitutive RNASEH1 exon 4/5 deletion

tag: age

value: 6 wk


title: S2 T4 rep2 traditional occupancy


title: WF-rep2

description: inducible wild type

source_name: WF


title: S2 T1 rep1 traditional occupancy


title: S2 T4 H3 ChIP rep2 100U MNase

source_name: UPR-induced S2 cells, T4 H3 ChIP 100U MNase

value: H3 (abcam, ab1791)


title: FC_6 wk-rep2

description: constitutive knockout (6 wk)


title: 2i/LIF – N2B27_BL6_Rep1

description: RNA-seq

source_name: 2i/LIF – N2B27_BL6_Rep1


title: FC_6 wk-rep1

description: constitutive knockout (6 wk)


title: S2 T4 H3 ChIP rep2 25U MNase

source_name: UPR-induced S2 cells, T4 H3 ChIP 25U MNase

value: S2 cell


title: FW_6 wk-rep3

description: constitutive wild type (6 wk)

source_name: FW_6 wk


title: 2i/LIF – N2B27_129_Rep2

description: RNA-seq


title: 2i/LIF – N2B27_129_Rep1

description: RNA-seq


title: S2 T4 H3 ChIP rep2 6.25U MNase

source_name: UPR-induced S2 cells, T4 H3 ChIP 6.25U MNase


title: 2i/LIF – N2B27_BL6_Rep2

description: RNA-seq

source_name: 2i/LIF – N2B27_BL6_Rep2


title: FW_6 wk-rep2

description: constitutive wild type (6 wk)


title: S2 T4 H3 ChIP rep2 1.5U MNase

source_name: UPR-induced S2 cells, T4 H3 ChIP 1.5U MNase


title: 2i/LIF – GMEM/10% serum_BL6_Rep2

description: RNA-seq

source_name: 2i/LIF – GMEM/10% serum_BL6_Rep2


title: 2i/LIF – GMEM/10% serum_BL6_Rep1

description: RNA-seq

source_name: 2i/LIF – GMEM/10% serum_BL6_Rep1


title: S2 T4 H3 ChIP rep1 100U MNase


title: S2 T4 H3 ChIP rep1 25U MNase


title: CF-rep1

description: inducible knockout

source_name: CF


title: FW_6 wk-rep1

description: constitutive wild type (6 wk)


title: FC_14 wk-rep3

description: constitutive knockout (14 wk)

source_name: FC_14 wk

value: 14 wk


title: 2i/LIF – GMEM/10% serum_129_Rep2

description: RNA-seq

source_name: 2i/LIF – GMEM/10% serum_129_Rep2


title: 2i/LIF – GMEM/10% serum_129_Rep1

description: RNA-seq

source_name: 2i/LIF – GMEM/10% serum_129_Rep1


title: FC_14 wk-rep2

description: constitutive knockout (14 wk)


title: S2 T4 H3 ChIP rep1 1.5U MNase

value: T4 (4 hours)


title: 2i/LIF – GMEM/10% KSR_BL6_Rep2

description: RNA-seq

source_name: 2i/LIF – GMEM/10% KSR_BL6_Rep2


title: FC_14 wk-rep1

description: constitutive knockout (14 wk)


title: 2i/LIF – GMEM/10% KSR_BL6_Rep1

description: RNA-seq

source_name: 2i/LIF – GMEM/10% KSR_BL6_Rep1


title: 2i/LIF – GMEM/10% KSR_129_Rep2

description: RNA-seq

source_name: 2i/LIF – GMEM/10% KSR_129_Rep2


title: FW_14 wk-rep2

description: constitutive wild type (14 wk)

source_name: FW_14 wk


title: S2 T1 H3 ChIP rep2 100U MNase

source_name: UPR-induced S2 cells, T1 H3 ChIP 100U MNase

value: T1 (1 hour)


title: S2 T1 H3 ChIP rep2 25U MNase

source_name: UPR-induced S2 cells, T1 H3 ChIP 25U MNase


title: S2 T1 H3 ChIP rep2 6.25U MNase

source_name: UPR-induced S2 cells, T1 H3 ChIP 6.25U MNase


title: FW_14 wk-rep1

description: constitutive wild type (14 wk)


title: 2i – N2B27_BL6_Rep2

description: RNA-seq

source_name: 2i – N2B27_BL6_Rep2


title: S2 T1 H3 ChIP rep2 1.5U MNase

source_name: UPR-induced S2 cells, T1 H3 ChIP 1.5U MNase


title: BCRABL_WT_SHP2_WT_REP_3

city: Toronto

first: Pamela

last: Ohashi

state: Ontario

address: 610 University Ave

country: Canada

institute: Princess Margaret Cancer Centre

data_processing:
Alignment was performed with Bowtie, using default parameters.
Gene expression levels were estimated using Cufflinks.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: FPKM

source_name: BCR-ABL WT (+/+)/SHP2 WT (+/+)_pre-B cells

extract_protocol: RNA (150 ng) from each sample was reverse-transcribed into cDNA using the Illumina TruSeq Stranded mRNA kit. cDNA libraries were size-indexed and validated using an Agilent Bioanalyzer, and concentrations were confirmed by qPCR. For the 16 samples, each of the 4 libraries (1 WT SHP2+/+, 1 WT SHP2-/-, 1 BCR-ABL1 SHP2+/+, and 1 BCR-ABL1 SHP2-/-) was loaded onto an Illumina cBot for cluster generation, and the flow cell was subjected to 100 cycles of paired-end sequencing on an Illumina HiSeq 2000.

value: BCR-ABL WT (+/+)/SHP2 WT (+/+)

value: pre-B cells


title: 2i – N2B27_BL6_Rep1

description: RNA-seq

source_name: 2i – N2B27_BL6_Rep1


title: 2i/LIF – GMEM/10% KSR_129_Rep1

description: RNA-seq

source_name: 2i/LIF – GMEM/10% KSR_129_Rep1


title: 2i – N2B27_129_Rep2

description: RNA-seq


title: BCRABL_WT_SHP2_WT_REP_2


title: S2 T1 H3 ChIP rep1 6.25U MNase


title: 2i – N2B27_129_Rep1

description: RNA-seq


title: S2 T1 H3 ChIP rep1 25U MNase


title: BCRABL_WT_SHP2_WT_REP_0


title: S2 T1 H3 ChIP rep1 1.5U MNase


title: BCRABL_WT_SHP2_WT_REP_1


title: S2 T4 H3 ChIP rep1 6.25U MNase

source_name: UPR-induced S2 cells, T4 H3 ChIP 6.25U MNase


title: BCRABL_WT_SHP2_MINUS_REP_3

source_name: BCR-ABL WT (+/+)/SHP2 NULL (-/-)_pre-B cells

value: BCR-ABL WT (+/+)/SHP2 NULL (-/-)


title: FW_14 wk-rep3

description: constitutive wild type (14 wk)


title: S2 T0 H3 ChIP rep2 25U MNase

source_name: UPR-induced S2 cells, T0 H3 ChIP 25U MNase

value: T0 (0 hours, control)


title: BCRABL_WT_SHP2_MINUS_REP_2


title: HSKO_3

city: Ithaca

first: Praveen

last: Sethupathy

address: T7 006 Vet Research Tower

institute: Cornell University

data_processing:
Sequencing quality was assess using fastqc/0.11.3
Reads were aligned using MapSplice/2.1.4 to the UCSC mm10 reference genome and bowtie index.
Alignments were quantified and used to generate gene level count tables using RSEM/1.2.9
Gene level counts were read into R/3.1.1. DESeq/1.16.0 was used to normalize counts and conduct differential gene expression analysis. A table of normalized counts for each gene and sample is included as processed data.
Genome_build: mm10
Supplementary_files_format_and_content: tab delimited text files include normalized (using DESeq) count values for each sample

source_name: liver

growth_protocol: Mice were fasted from 9 am to 1 pm and then sacrificed for liver harvest and RNA extraction.

extract_protocol:
RNA was extracted from mouse livers using the Norgen Total RNA Purification kit.
Libraries were prepared using TruSeq Stranded mRNA protocol per manufacturer's instructions by the UNC High Throughput Sequencing Core Facility. Paired end sequencing was performed on an Illumina HiSeq 2500.

value: ABCA1 deletion


title: BCRABL_WT_SHP2_MINUS_REP_1


title: HSKO_1


title: S2 T1 H3 ChIP rep1 100U MNase


title: S2 T0 H3 ChIP rep2 1.5U MNase

source_name: UPR-induced S2 cells, T0 H3 ChIP 1.5U MNase


title: BCRABL_WT_SHP2_MINUS_REP_0


title: WT_2

value: wildtype


title: WT_3

value: liver


title: S2 T0 H3 ChIP rep2 6.25U MNase

source_name: UPR-induced S2 cells, T0 H3 ChIP 6.25U MNase


title: BCRABL_MINUS_SHP2_WT_REP_3

source_name: BCR-ABL NULL (-/-)/SHP2 WT (+/+)_pre-B cells

value: BCR-ABL NULL (-/-)/SHP2 WT (+/+)


title: WT_1


title: S2 T0 H3 ChIP rep1 25U MNase


title: BCRABL_MINUS_SHP2_WT_REP_2


title: 4Cseq_HYa_rep2

description: HindIII-digested gDNA

data_processing:
Library strategy: 4C-Seq
Basecalls performed using CASAVA.
Demultiplexing performed using fastx_barcode_splitter, and viewpoint sequences were trimmed from 5' end of reads using fastx_trimmer.
Alignment to genome was performed using Bowtie with parameters -v 2 -m 1 --all --best --strata
Mapped reads were processed using samtools and bedgraphs generated using GenomeCoverageBed.
Reads were normalized to mapped reads per million within the Tcra-Tcrd locus and visualized using UCSC genome browser.
Genome_build: mm9, with endogenous Tcra-Tcrd replaced by strain 129 sequence.
Supplementary_files_format_and_content: .bedgraph files with read counts normalized to reads per million reads within the Tcra-Tcrd locus.

source_name: DP thymocytes

growth_protocol: 3-week-old mice were injected i.p. with 150 micrograms of anti-CD3-epsilon antibody 10 days prior to sacrifice.

extract_protocol: Thymocytes were crosslinked and chromatin was subjected to HindIII restriction digest. Sticky ends were re-ligated using T4 DNA ligase and DNA was purified from nuclear lysates. DNA was subsequently digested with BfaI and re-ligated with T4 DNA ligase to make 4C library templates.

value: Rag2-/- x HYa

value: thymus

value: DP thymocytes


title: S2 T0 H3 ChIP rep1 6.25U MNase


title: S2 T0 H3 ChIP rep1 1.5U MNase


title: 4Cseq_HYa_rep1

description: HindIII-digested gDNA


title: BCRABL_MINUS_SHP2_WT_REP_0


title: HSKO_2


title: BCRABL_MINUS_SHP2_MINUS_REP_2

source_name: BCR-ABL NULL (-/-)/SHP2 NULL (-/-)_pre-B cells

value: BCR-ABL NULL (-/-)/SHP2 NULL (-/-)


title: 4Cseq_Rag2_rep2

description: HindIII-digested gDNA

value: Rag2-/- x strain 129


title: S2 T0 H3 ChIP rep2 100U MNase

source_name: UPR-induced S2 cells, T0 H3 ChIP 100U MNase

value: S2 cell

value: H3 (abcam, ab1791)


title: Stephensi_MG_RNA-seq

city: Bangalore

first: T.S. Keshava

last: Prasad

state: Please select one below:

address: 7th floor, Discoverer block, ITPL

country: India

institute: Institute of bioinformatics

data_processing:
Basecalls were performed using Illumina CASAVA v1.8
Sequenced reads were trimmed for adapter sequences and quality filtered using fqtrim v0.9.4. The trimmed reads were aligned to An. stephensi genome build (version 2) (ASTEI2) from VectorBase using HISAT2
Aligned reads were assembled into transcripts against the An. stephensi AsteI2.2 gene annotations, as reference, using the StringTie (version 1.2.1) assembler. The parameters include -a 10 -m 200 -j 5 -c 2.5  The assemblies on individual tissues were merged using the StringTie merge option.
The transcript assemblies were annotated against the VectorBase gene annotations AsteI2.2 using gffcompare and the differential expression was assessed using cuffdiff.
Genome_build: ASTEI2
Supplementary_files_format_and_content: tab-delimited text files including FPKM values for each tissue

organism: Anopheles stephensi

source_name: Midgut

growth_protocol: Tissues were dissected from sugar fed female An. stephensi adult mosquitoes grown in the lab under ambient conditions (humidity 70+5%, temperature 27±2°C and photoperiod:scotoperiod of 12:12 h)

extract_protocol:
Total RNA was extracted  using miRNeasy kit (Qiagen) according to the manufacturer’s protocol after homogenization of tissues using MINILYS benchtop homogenizer and Precelly Lysis Kit (PEQLAB).
Indexed paired-end libraries were prepared according to the manufacturer's instructions as per Illumina TruSeq RNA Sample Preparation Kit v3.

value: Midgut

value: Adult

tag: Sex

value: Female


title: sickle R. solani inoculated treated 7 dpi rep 3

source_name: Seedling root tissue

value: sickle mutant (ethylene insensitive, point mutation in A17)

value: 7 days

value: Highly susceptible to R. solani AG8


title: Stephensi_MT_RNA-seq

source_name: Malpighian tubules

value: Malpighian tubules


title: BCRABL_MINUS_SHP2_MINUS_REP_1

value: pre-B cells


title: Stephensi_Ov_RNA-seq

source_name: Ovaries

value: Ovaries


title: His6-ctUtp4

city: Heidelberg

first: Ed

last: Hurt

address: Im Neuenheimer Feld 328

department: BZH

country: Germany

institute: Biochemie Zentrum Heidelberg

data_processing:
Duplicate reads were filtered by script In pyCRAC program
Reads were mapped to the 5’- ETS sequence of Chaetomium thermophilum using the Novoalign software.
Coverage and mutation hotspots were calculated with pyCRAC.
Genome_build: Ct. 5'-ETS (ct5ETS.fasta)
Supplementary_files_format_and_content: pyCRAC pileup format for Ct. 5'-ETS

organism:
Saccharomyces cerevisiae
Thermochaetoides thermophila

source_name: In vitro-UV CRAC from His6-Utp4 expressing cells

growth_protocol: Yeast cells were grown in raffinose (SRC-Trp-Leu- Ura-His)-containing medium to an OD600nm of ~2.0, before GAL promoter was induced by addition of 2-fold concentrated galactose-containing medium (2x YPG) at a ratio of 1:1.

extract_protocol: For extraction and library preparation protocol please refer to the Materials and Methods section.

treatment_protocol: Cells were harvested after induction for 5 hours by centrifugation at 4000 ×g, washed with dH2O, and frozen in liquid nitrogen.

tag: strain background

value: yeast strain W303

value: expressing Ct. 5'-ETS and co-expressing six Ct. UTP-A factors (His6-Utp4, Utp5, Utp8, proteinA-TEV-Utp10, Flag-Utp15, and Utp17)


title: No tag control

source_name: In vitro-UV CRAC

value: expressing Ct. 5'-ETS and co-expressing six Ct. UTP-A factors (Utp4, Utp5, Utp8, proteinA-TEV-Utp10, Flag-Utp15, and Utp17)


title: S2 T0 H3 ChIP rep1 100U MNase

value: T0 (0 hours, control)

tag: chip antibody


title: Stephensi_FB_RNA-seq

source_name: Fat body

value: Fat body


title: BCRABL_MINUS_SHP2_MINUS_REP_0

tag: genotype/variation

value: BCR-ABL NULL (-/-)/SHP2 NULL (-/-)


title: treated rep1_RNA-seq

status: Public on Jun 05 2017

city: Nanjing

first: Wei

last: Dai

state: Jiangsu

address: Sipailou 2#,Nanjing, Jiangsu Province, P. R. China

institute: Southeast University

data_processing:
Illumina HiSeq 2500 reads were checked for each sample using FastQC v0.10.1
High-quality reads were aligned to UCSC hg19 into BAM files with HISAT and Htseq
Aligned reads were analyzed with Cufflinks and Cuffdiff, and RPKM counts were calculated
Differentially expressed genes were calculated with DEGseq and DEseq2
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files

source_name: human hepatocellular carcinoma HepG2_TNFα-treated

growth_protocol: HepG2 cells were cultured in DMEM medium, complemented with 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin.

extract_protocol:
Total RNA was extracted from uninduced, TNFα-induced and siRNA and TNFα dually treated HepG2 cells, respectively, with Trizol® reagent (Invitrogen)
1 μg of total RNA was subjected to library synthesis using the TrueSeq RNA Sample Preparation Kit (Illumina)

treatment_protocol: HepG2 cells were incubated at 37°C in 5% CO2, when grown to a density of 1‒5×10^6/mL cells were treated with 10 ng/mL of TNFα in serum-free DMEM at 37 °C for 1 h. For siRNA transfection, when grown to a density of 1‒5×10^6/mL, HepG2 cells were firstly transfected with NF-κB RelA siRNA at a concentration of 30 nM for 4 h in serum-free DMEM using Lipofectamine 2000. After culturing with fresh media containing 10% FBS for 48 h, HepG2 cells were treated with TNFα at the concentration of 10 ng/mL in serum-free DMEM at 37 °C for 1 h. Two biological replicates were grown in separate batches and at separate times.

tag: cell line

value: primary epithelial cells

value: liver


title: treated rep2_RNA-seq


title: interference rep1_RNA-seq

source_name: human hepatocellular carcinoma HepG2_NF-κB RelA siRNA_TNFα-treated


title: interference rep2_RNA-seq


title: untreated rep1_RNA-seq

source_name: human hepatocellular carcinoma HepG2_untreated


title: untreated rep2_RNA-seq


title: BCRABL_MINUS_SHP2_WT_REP_1

value: BCR-ABL NULL (-/-)/SHP2 WT (+/+)


title: IgG untreated

data_processing:
Sequencing raw data was converted to fastq format and controlled for general quality features using FastQC.
ChIP-seq reads were aligned to unique position on precompiled hg19 reference index by Bowtie with the default setting allowing up to 2 mismatches.
Peaks were identified by MACS v2.1.0 at default settings by comparing specific ChIP-seq reads above with unspecific control reads derived from sequencing matching IgG samples. Peaks were called at a Poisson p-value < 10-4 and an FDR < 5% and were used for intersection analysis to determine the overlap in pair wise comparisons.
All operations on genomic intervals like measuring and counting overlap as well as measuring distances between genomic features were performed using R's GenomicRanges package. In order to identify differentially bound regions all binding regions determined for a given factor under different conditions were collapsed and the read numbers mapping to these collapsed intervals were extracted.
In case the overlap of multiple binding factors was studied at once the multiinter function of the BedTools suite was used with the cluster option turned on.
A peak is assigned to a gene if it locates in the gene region of the gene with HOMER perl script annotatePeaks.pl.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig file

extract_protocol:
5‒10 ng of immunoprecipitated DNA were prepared ChIP-Seq libraries.
ChIP-Seq libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Set 2) (New England Biolabs).

treatment_protocol: HepG2 cells were incubated at 37°C in 5% CO2, when grown to a density of 1‒5×10^6/mL cells were treated with 10 ng/mL of TNFα in serum-free DMEM at 37 °C for 1 h. Cells were collected by pancreatic enzymes digestion and fixed in vivo with 1% formaldehyde for 10 min at room temperature, followed with 1.25 M glycine for 5 min at room temperature to stop the cross-linking. Then, cells were centrifuged at 1,610g (10 min, 4°C), washed twice in cold PBS containing 1 mM PMSF and 1 mM protease inhibitor cocktail (PIC). Cells were swelled for 20 min on ice in 0.5 mL ChIP cell lysis buffer and centrifuged at 587g (10 min, 4°C). Nuclear pellets were lysed on ice in 0.5 mL ChIP nuclei lysis buffer. The chromatin was sheared by sonication (40×30s on/30s off; Branson, USA) and lysates cleared by centrifugation at 16,100g (15 min, 4°C). Supernatants were collected and stored in aliquots at –80°C for subsequent ChIP. DNA was purified using QIA quick PCR purification kit.

value: normal rabbit IgG (Santa Cruz, sc-2027)


title: IgG treated


title: BCRABL_MINUS_SHP2_MINUS_REP_3


title: p65 untreated rep2

value: NF-κB (Santa Cruz, C-20, sc-372)


title: p65 treated rep1


title: 4Cseq_Rag2_rep1

institute: Duke University

description: HindIII-digested gDNA

value: Rag2-/- x strain 129

value: thymus

value: DP thymocytes


title: p65 treated rep2


title: p65 untreated rep1


title: vehicle, 60 min, rep 5-mRNAseq

city: Berkeley

first: Jamie

last: Cate

address: 748 Stanley Hall

institute: University of California

data_processing:
Adapter sequences (CTGTAGGCACCATCAAT) were trimmed and reads were filtered for minimum length using FASTX-clipper version 0.0.13
Ribosomal RNA sequences were computationally removed by aligning the reads to a set of human rRNA sequences with Bowtie version 1.1.2
Nonribosomal sequences from samples 24 and 26 were pooled were pseudoaligned to a reference consiting of all transcripts annotated as "Known" and "Protien coding" in the ENSEMBL GRCh38 annotation using Kallisto version 0.42.5 to determine the relative abundance of the transcript isoforms. This was done with python version 3.5.1 . Python code used is Additional_file_S2.py
A custom reference transcriptome consisting of the most abundant transcript isoform from each gene was generated using python version 3.5.1. Python code used is "Additional_file_S4.py" and "Additional_file_S5.py"
Nonribosomal sequences were aligned with the custom transcriptome using Bowtie version 1.1.2
Reads mapping to more then five locations in the custom transcriptome were filtered out using python version 2.6.6. Python code used is "Additional_file_S6.py"
Readmaps indicating the number of reads alining to each position of the custom transcriptome were genenerated using python 2.6.6. The python code used is "Additional_file_S7.py"
Metareadmaps" showing the average distribution of reads relative to the start and stop codons for all genes were generated using python version 2.6.6 These maps were used to determine the distance between the 5' end of read alingments and the ribosomal P-site. The python code used for this step is "Additional_file_S8.py
The readmaps were converted from nucleotide-based to codon-based using python 2.6.6. The python code used is "Additional_file_S9.py"
The readmaps were normalized from raw reads to reads per million using python 2.6.6. The python code used is "Additional_file_S10.py"
Cumulative fractional readplots, Dmax values and Dmax positions were calculated using python 2.6.6. The output of "Additional_file_S9.py" is used for this step. The python code for this step is "Additional_file_S11.py"
Dmax values were Z-score transformed as described in Andreev et al. (2015) Genome Biology 16:90 using python verson 2.6.6 The python code used for this step is "Additional_file_S17.py"
For genes with a Dmax Zscore > 2 reads mapping to the CDS 3' to the position of Dmax were extracted for DeSeq analysis. For Dmax Z-score ≤ 2, reads mapping 3' to codon 50 were extracted. In all cases, the 10 3'-most codons were omitted. The python code used for this step is "Additional_file_S12.py"
The extracted reads were used for differential expression analysis with DeSeq1. Changes in the number of reads extracted in the previous step were used as a basis for determining compound-induced changes in expression levels.

ADDITIONAL ANALYSIS:
For standard differential expression analysis, raw read counts were extracted from the output of "Additional_file_S7.py" python code "Additional_file_S13.py" for mRNA-seq datasets or "Additional_file_S14.py" for riboseq datasets. The output was used as input for DeSeq for standard differential expression analysis.
Z-score transformations analysis (Andreev et al. (2015) Genome Biology 16:90) was done on raw alignment counts and tranlational efficiency values using python version 2.6.6, "Additional_file_S15.py", "Additional_file_S16.py" and "Additional_file_S18.py"
Centers of density or the mean positions of reads along each CDS, changes in the centers of density and Z-score transformations of those changes were calculated using python version 2.6.6. The code is "Additional_file_S19.py"
Potential AUG-initiating uORFs were identified and the number of reads aligning to those uORFs were extracted using python version 2.6.6 The code is "Additional_file_S20.py" and "Additional_file_S21.py"
Genome_build: GRCh38

Supplementary_files_format_and_content:
*codonmap.txt files are plain text files with three columns, the first is the transcript accession number, the second is the codon, relative to start, the third is the number of reads that align to that position based on the infered location of the ribosome P-site.
Deseq_poststall_analysis.xlsx is an Excel spreadsheet with Differential expression analysis that uses the Dmax approach to predict potential stall sites and use only alignments mapping 3' to thos sites.
Deseq_basic_analysis.xlsx is an Excel spreadsheet with basic differential expression analyses done with DeSeq
Z-score_analyses.xlsx is an Excel spreadsheet with Z-score analyses of raw readcounts as described in Andreev et al. (2015) Genome Biology 16:90
Center_of_densities.xlsx is an Excel spreadsheet with Center of density analysis as described in Andreev et al. (2015) Genome Biology 16:90
Raw_read_counts.xlsx is an Excel speadsheet summarizing raw alignment counts.
Additional_files_S1-S21.txt is an Text file containing the custom python code used to process the data.
GRCh38_known_protein_coding_cDNAs.fa is the Multifasta file with reference transcriptome used for the initial Kallisto psudoaligments.
GRCh38_Huh7_most_abundant_isoforms.fa is the Multifasta file with reference transcriptome of only the most abundant splice isoform from each locus, used in the bowtie alignments.

source_name: Huh7_vehicle_60 min_RNA-seq

growth_protocol: Huh7 cells were maintained in RPMI ((Life Technologies 11875-093) supplemented with 10% FBS (Sigma F4135) 1% Penicillin/Streptomycin (Life Technologies 15410-112) and 4 mM Glutamax (Life Technologies 35050-061) at 37 ˚C under a 5% CO2 atmosphere.   The cells were tested for mycoplasma and using the Gen-Probe Mycoplasma Tissue Culture NI (MTC-IN) Rapid Detection method.

extract_protocol:
The cells were harvested by aspiration of media and immediate addition of ice-cold PBS containing 100 µg/mL cycloheximide.  The PBS plus cycloheximide was aspirated with a vacuum aspirator and 400 µL lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 100 µg/mL cycloheximide, 1% Triton X-100 and 25 U/mL DNase I (Promega)) was added.
Cells were lysed and ribosome footprint or fragmented RNA libraries were generated as described in Ingolia et al. Curr Protoc Mol Biol Chapter 4, Unit 4 18 (2013).

treatment_protocol: Huh7 cells were grown in 10 cm plates to a confluence of 50-80%.  The cells were always passaged into fresh media within 24 hours of the experiment. The media was replaced with fresh prewarmed media containing 1.5 µM or 0.3 µM PF-06446846 or vehicle (0.5% DMSO) and the plates were immediately returned to the incubator for 10 or 60 minutes as indicated.

tag: treatment time

value: 60 min

tag: library type

value: polyA-seq


title: PF-06446846, 60 min, rep 5 -mRNA-seq

data_processing:
library strategy: Ribo-seq
Adapter sequences (CTGTAGGCACCATCAAT) were trimmed and reads were filtered for minimum length using FASTX-clipper version 0.0.13
Ribosomal RNA sequences were computationally removed by aligning the reads to a set of human rRNA sequences with Bowtie version 1.1.2
Nonribosomal sequences from samples 24 and 26 were pooled were pseudoaligned to a reference consiting of all transcripts annotated as "Known" and "Protien coding" in the ENSEMBL GRCh38 annotation using Kallisto version 0.42.5 to determine the relative abundance of the transcript isoforms. This was done with python version 3.5.1 . Python code used is Additional_file_S2.py
A custom reference transcriptome consisting of the most abundant transcript isoform from each gene was generated using python version 3.5.1. Python code used is "Additional_file_S4.py" and "Additional_file_S5.py"
Nonribosomal sequences were aligned with the custom transcriptome using Bowtie version 1.1.2
Reads mapping to more then five locations in the custom transcriptome were filtered out using python version 2.6.6. Python code used is "Additional_file_S6.py"
Readmaps indicating the number of reads alining to each position of the custom transcriptome were genenerated using python 2.6.6. The python code used is "Additional_file_S7.py"
Metareadmaps" showing the average distribution of reads relative to the start and stop codons for all genes were generated using python version 2.6.6 These maps were used to determine the distance between the 5' end of read alingments and the ribosomal P-site. The python code used for this step is "Additional_file_S8.py
The readmaps were converted from nucleotide-based to codon-based using python 2.6.6. The python code used is "Additional_file_S9.py"
The readmaps were normalized from raw reads to reads per million using python 2.6.6. The python code used is "Additional_file_S10.py"
Cumulative fractional readplots, Dmax values and Dmax positions were calculated using python 2.6.6. The output of "Additional_file_S9.py" is used for this step. The python code for this step is "Additional_file_S11.py"
Dmax values were Z-score transformed as described in Andreev et al. (2015) Genome Biology 16:90 using python verson 2.6.6 The python code used for this step is "Additional_file_S17.py"
For genes with a Dmax Zscore > 2 reads mapping to the CDS 3' to the position of Dmax were extracted for DeSeq analysis. For Dmax Z-score ≤ 2, reads mapping 3' to codon 50 were extracted. In all cases, the 10 3'-most codons were omitted. The python code used for this step is "Additional_file_S12.py"
The extracted reads were used for differential expression analysis with DeSeq1. Changes in the number of reads extracted in the previous step were used as a basis for determining compound-induced changes in expression levels.
ADDITIONAL ANALYSIS:
For standard differential expression analysis, raw read counts were extracted from the output of "Additional_file_S7.py" python code "Additional_file_S13.py" for mRNA-seq datasets or "Additional_file_S14.py" for riboseq datasets. The output was used as input for DeSeq for standard differential expression analysis.
Z-score transformations analysis (Andreev et al. (2015) Genome Biology 16:90) was done on raw alignment counts and tranlational efficiency values using python version 2.6.6, "Additional_file_S15.py", "Additional_file_S16.py" and "Additional_file_S18.py"
Centers of density or the mean positions of reads along each CDS, changes in the centers of density and Z-score transformations of those changes were calculated using python version 2.6.6. The code is "Additional_file_S19.py"
Potential AUG-initiating uORFs were identified and the number of reads aligning to those uORFs were extracted using python version 2.6.6 The code is "Additional_file_S20.py" and "Additional_file_S21.py"
Genome_build: GRCh38
Supplementary_files_format_and_content:
*codonmap.txt files are plain text files with three columns, the first is the transcript accession number, the second is the codon, relative to start, the third is the number of reads that align to that position based on the infered location of the ribosome P-site.
Deseq_poststall_analysis.xlsx is an Excel spreadsheet with Differential expression analysis that uses the Dmax approach to predict potential stall sites and use only alignments mapping 3' to thos sites.
Deseq_basic_analysis.xlsx is an Excel spreadsheet with basic differential expression analyses done with DeSeq
Z-score_analyses.xlsx is an Excel spreadsheet with Z-score analyses of raw readcounts as described in Andreev et al. (2015) Genome Biology 16:90
Center_of_densities.xlsx is an Excel spreadsheet with Center of density analysis as described in Andreev et al. (2015) Genome Biology 16:90
Raw_read_counts.xlsx is an Excel speadsheet summarizing raw alignment counts.
Additional_files_S1-S21.txt is an Text file containing the custom python code used to process the data.
GRCh38_known_protein_coding_cDNAs.fa is the Multifasta file with reference transcriptome used for the initial Kallisto psudoaligments.
GRCh38_Huh7_most_abundant_isoforms.fa is the Multifasta file with reference transcriptome of only the most abundant splice isoform from each locus, used in the bowtie alignments.

value: mRNA-seq


title: vehicle, 60 min, rep 4-mRNAseq