title: RNAseq_D7P14_TOX_Over_TerminallyExhausted_3

data_processing:
Raw sequencing data were processed with CASAVA 1.8.2 to generate FastQ files. Reads of sequentially varying length were mapped to the mouse genome mm10 using TopHat 2.1.0.  RPKM calculated with Partek.
Genome_build: mm10
Supplementary_files_format_and_content: text file with RPKM

extract_protocol:
cell isolation protocol: Cells from spleen were obtained by mechanical disruption. Isolated cells were further sorted based on their surface markers.
Lymphocytic choriomeningitis virus infection protocol: For acute viral infections, mice were intravenously (i.v.) injected with 2×10^5 plaque-forming unit (PFU) LCMV Armstrong. For chronic viral infections, mice were intravenously (i.v.) injected with 2×10^6 PFU LCMV clone 13.
50,000 P14 cells were sorted and resuspended in Qiazol (Qiagen). Total RNA was extracted with miRNeasy Mini kit (Qiagen). mRNA was selected using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). RNAseq libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for (NEB) according to manufacturer’s instruction.
The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on HiSeq 3000  according to the manufacturer's protocols.

tag: genotype/variation

value: TOX overexpression

tag: culture condition


title: PF052-95

status: Public on Apr 16 2019

type: SRA

city: New Delhi

first: Shashank

last: Jatav

state: Delhi

address: 212, C/o Brijendar Pawar, Near Dalip Singh Chowk, Shahpur Jat

country: India

institute: Elucidata

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
C08_S5_L001_R1_001.fastq.gz
C08_S5_L002_R1_001.fastq.gz
C08_S5_L003_R1_001.fastq.gz
C08_S5_L004_R1_001.fastq.gz

library_source: transcriptomic

data_processing:
Transcriptional data was aligned using STAR (https://github.com/alexdobin/STAR) aligner.
Gene counts from resulting bam files were generated using featurecounts using GTF file from UCSC (http://bioinf.wehi.edu.au/featureCounts/).
Genome_build: hg38
Supplementary_files_format_and_content: comma separated text files include raw counts for each Sample, the first column contains the geneid, the raw counts start from column 7, the format followed is same featurecounts' output

molecule: polyA RNA

organism: Homo sapiens

source_name: Human T cells

growth_protocol: Blood was collected from healthy human patients in accordance with IRB standards. CD4+ T cells were purified using RosetteSep isolation kit (StemCell) as per manufacturer’s protocol. Cells were cultured in RPMI supplemented with 10% heat inactivated FBS, HEPES, penicillin/streptomycin, non-essential amino acids, glutamax, and sodium pyruvate. Cells were initially activated for 72 hours on plate-bound CD3 (200 ng/mL; OKT3; BioXCell) and CD28 (200 ng/mL; 9.3; BioXCell) on goat-anti-mouse (10 µg/mL; Jackson Immuno Research Labs) pre-coated high- binding plates (Corning Costar). After 72 hours, cells were removed from plate activation and were expanded until Day 8-12, maintained at ~1X106 cells/mL by supplementing with fresh media as required. Cells were then washed with PBS and either rested or re-activated at 0.5-1x106 cells/mL in fresh media with plate-bound CD3/CD28 (100 ng/mL each) or with soluble CD3/CD28 Immunocult (1:100 dilution; StemCell).

extract_protocol:
5x10^6 cells were collected at the indicated time-points, lysed with RNeasy (Qiagen) kit, and purified according to manufacturer?s protocol. RNA quantity was assessed using the QIAxpert and RNA quality was assessed using the Agilent 4200 TapeStation.
Each sample is represented by 4 technical replicates. Technical replicates were produced by sequencing the same library in 4 separate flowcell lanes.
RNA library preparation was performed using Illumina Stranded mRNA library preparation kit and sequencing was performed using NextSeq High Output Run with single end read 1x75bp.

tag: cell type

value: Primary human CD4+ T cells

tag: re-activation

value: Plate bound CD3/CD28 (100ng/mL)

tag: reactivation hours

tag: knockdown

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run

tag: sample name (randomized)

tag: experiment number


title: RNAseq_D7P14_TOX_Over_ProgenitorLike_3

city: Bethesda

first: Tuoqi

last: Wu

state: MD

address: 49 Convent Dr.

country: USA

institute: National Institute of Allergy and Infectious Diseases

organism: Mus musculus

source_name: CD8 P14

tag: strain

tag: tissue

value: Spleen

tag: surface marker

value: ex vivo


title: RNAseq_D7P14_TOX_Over_TerminallyExhausted_1


title: PF052-97

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D06_S7_L001_R1_001.fastq.gz
D06_S7_L002_R1_001.fastq.gz
D06_S7_L003_R1_001.fastq.gz
D06_S7_L004_R1_001.fastq.gz

value: shmTOR


title: cMO_exp2_st10p_polyA-RNA_rep1

city: London

first: George

middle: E.

last: Gentsch

state: Greater London

address: 1 Midland Road

department: Developmental Biology Laboratory

country: United Kingdom

institute: The Francis Crick Institute

description: standard control morphant, stage 10+ (early gastrula), experiment 2, poly(A)+ RNA, biological replicate 1

data_processing:
Bases were called using Casava version 1.8.2
ChIP-Seq: Single reads of maximal 50 bases were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Processed reads were aligned to the X. tropicalis genome assembly v7.1 (and v9.1 for some ChIP-Seq) running Bowtie2 v2.2.9 with default settings. Alignments were converted to HOMER’s tag density format with redundant reads being removed (makeTagDirectory -single -tbp 1 -unique -mapq 10 -fragLength 175 -totalReads all). For all genomic regions except for the mir427 gene cluster, only uniquely aligned reads (i.e. MAPQ ≥10) were processed. The highly repetitive mir427 locus was profiled with all reads only best aligning up to 80 times (-k 80) to this region. In this case the mass of each read was normalised to the number of alignments. We pooled all input alignments from various developmental stages. This created a comprehensive mappability profile that covered ~400 million unique base pair positions. All chromatin profiles were position-adjusted and normalised to the effective total of 1 million aligned reads including multimappers (counts per million aligned reads, CPM). Transcription factor binding sites were identified using the following or otherwise default parameters of HOMER’s calling of ChIP-mediated read enrichments (hereafter called peaks): findpeaks -style factor -minDist 175 -fragLength 175 -inputFragLength 175 -fdr 0.001 -gsize 1.435e9 -F 3 -L 1 -C 0.97. This means that both ChIP and input alignments were extended 3’ to 175 bp for the detection of significant (0.1% FDR) peaks being separated by ≥175 bp. The effective size of the X. tropicalis genome assembly v7.1 was set to 1.435 billion bp, an estimate obtained from the mappability profile. These peaks showed equal or higher tag density than the surrounding 10 kb, ≥3-fold more tags than the input and ≥0.97 unique tag positions relative to the expected number of tags. To detect focal RNAPII recruitment to putative cis-regulatory elements and avoid calling peaks within broad regions of RNAPII elongation, the threshold of focal ratio and local enrichment within 10 kb was elevated to 0.6 and 3 (-L 3), respectively. To further eliminate any false positive peaks, we removed any peaks with <0.5 (transcription factors including signal mediators) or <1 (RNAPII) CPM and those falling into blacklisted regions showing equivocal mappability due to genome assembly errors, gaps or simple/tandem repeats. Regions of equivocal mappability were identified by a 2-fold lower (poor) or 3-fold higher (excessive) read coverage than the average detected in 400-bp windows sliding at 200-bp intervals through normalised ChIP input and DNase-digested naked genomic DNA. All identified regions ≤800 bp apart were subsequently merged. Gap coordinates were obtained from the Francis Crick UCSC genome browser (http://genomes.crick.ac.uk). Simple repeats were masked with RepeatMasker v4.0.6 using the crossmatch search engine v1.090518 and the following settings: RepeatMasker -species "xenopus silurana tropicalis" -s -xsmall. Tandem repeats were masked with Kent’s trfBig wrapper script of the Tandem Repeat Finder v4.09 using the following settings: weight for match, 2; weight for mismatch, 7; delta, 7; matching probability, 80; indel probability, 10; minimal alignment score, 50; maximum period size, 2,000; and longest tandem repeat array (-l), 2 [million bp].
DNase-Seq: Single and paired-end reads of maximal 50 bases were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Processed reads were aligned to the X. tropicalis genome v7.1 and v9.1 using Bowtie2 v2.2.9 with default settings apart from -X (fragment length), which was reduced to 250 bp for paired-end reads. Alignments were sorted by genomic coordinates and only those with a quality score of ≥10 were retained using samtools v1.3.1. Duplicates were removed using Picard (MarkDuplicates). Paired-end alignments were dissociated using hex flags (-f 0x40 or 0x80) of samtools view. Single alignments were converted to HOMER’s tag density format (makeTagDirectory -single -unique -fragLength 100 -totalReads all). DNase hypersensitive sites (DHSs) were identified using the following or otherwise default parameters of HOMER’s peak calling: findpeaks -style factor -minDist 100 -fragLength 100 -inputFragLength 100 -fdr 0.001 -gsize 1.435e9 -F 3 -L 1 -C 0.97. This means that alignments of DNase-digested chromatin fragments (or naked genomic DNA fragments considered here as ‘input’) were extended 3’ by 100 bp from the DNase cleavage site to detect significant (0.1% FDR) DNase-mediated read enrichments (hereafter called peaks) being separated by ≥100 bp. The effective size of the X. tropicalis genome assembly v7.1 was set to 1.435 billion bp, an estimate obtained from the mappability profile (see ‘ChIP-Seq’ section). These peaks showed equal or higher tag density than the surrounding 10 kb, ≥3-fold more tags than the input and ≥0.97 unique tag positions relative to the expected number of tags. Peaks falling into blacklisted regions (see ‘ChIP-Seq’ section) were removed. DNase-probed chromatin accessibility profiles were position-adjusted and normalised to the effective total of 1 million aligned reads including multimappers.
Next-generation capture-C: Paired-end reads were processed using trim_galore v0.4.2 to trim off low-quality bases and adapter contamination from the 3’ end. Only complete mate pairs were processed further to reconstruct single reads from overlapping paired-end sequences using FLASH v1.2.11 with interleaved output settings for non-extended reads (flash --interleaved-output --max-overlap 150). FLASH reads were split in silico at DpnII restriction sites using a designated perl script (dpnII2E.pl, https://github.com/Hughes-Genome-Group/captureC/releases) before aligning them to the X. tropicalis genome assembly 7.1 using Bowtie2 v2.2.9. The alignment was run with default settings and one thread only to maintain the order of reads. The view function of samtools v1.3.1 was used to retain alignments with a quality score of ≥10. The alignments were analysed further using a suite of perl scripts (https://github.com/Hughes-Genome-Group/captureC/releases) modified to process both chromosome and scaffold coordinates. Viewpoint coordinates included a 1-kb proximity exclusion range. Restriction fragments were classified as capture, proximity-exclusion or reporter. PCR duplicates were removed. The interaction map was based on the number of unique paired-end reads per restriction fragment. Windows of 2 kb incrementing by 200 bp were used to consolidate interactions, which were normalised to 10,000 interactions per viewpoint. Significant chromatin conformation changes were identified using the R package DESeq2 v1.14.1.
RNase-Seq: Paired-end reads were aligned to the X. tropicalis genome assembly v7.1 using STAR v2.5.3a with default settings and a revised version of gene models v7.2 to improve mapping accuracy across splice junctions. The alignments were sorted by read name using the sort function of samtools v1.3.1. Exon and intron counts (-t 'exon;intron') were extracted from unstranded (-s 0) alignment files using VERSE v0.1.5 in featureCounts (default) mode (-z 0). Intron coordinates were adjusted to exclude any overlap with exon annotation. Differential expression analysis was performed with both raw exon and intron counts excluding those belonging to ribosomal and mitochondrial RNA using the R package DESeq2 v1.14.1. In an effort to find genes with consistent fold changes over time, p-values were generated according to a likelihood ratio test reflecting the probability of rejecting the reduced (~ developmental stage) over the full (~ developmental stage + condition) model. Resulting p-values were adjusted to obtain false discovery rates (FDR) according to the Benjamini-H

organism: Xenopus tropicalis

source_name: whole embryo

growth_protocol: Embryos were grown to the indicated developmental stage in 5% MMR at 25ºC.

extract_protocol:
Chromatin immunoprecipitation (ChIP): Briefly, dejellied X. tropicalis embryos were treated with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 15-45 min at room temperature to cross-link chromatin proteins to nearby genomic DNA. Duration of fixation was determined empirically and depended mainly on the developmental stage and antibody epitopes as described in Gentsch and Smith (2017). Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos. Methods in Molecular Biology, 1507, 23-42. Fixation was terminated by rinsing embryos three times with ice-cold 1% MMR. If required, post-fixation embryos were dissected to select specific anatomical regions in ice-cold 1% MMR. Fixed embryos were homogenized in CEWB1 (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS) supplemented with 0.5 mM DTT, protease inhibitors and, if using phospho-specific antibodies, phosphatase blockers (0.5 mM orthovanadate and 2.5 mM NaF). To solubilise yolk platelets and separate them from the nuclei, the homogenate was left on ice for 5 min and then centrifuged (1000 g) for 5 min at 4ºC. Homogenization and centrifugation was repeated once before resuspending the nuclei containing pellet in 1-3 ml CEWB1. Nuclear chromatin was solubilized and fragmented by isothermal focused or microtip-mediated sonication. The solution of fragmented chromatin was cleared by centrifuging (15,000 g) for 5 min at 4ºC. If required, ~1% of the cleared chromatin extract was set aside for the input sample (negative control for ChIP). ChIP-grade antibodies were used to recognize specific chromatin features and to enrich these by coupling the antibody-chromatin complex to protein G magnetic beads and extensive washing. These steps were carried out at 4ºC. The beads were washed twice in CEWB1, twice in WB2 (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS), twice in WB3 (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Igepal CA-630 and 1% sodium deoxycholate) and once in TEN (10 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA). ChIP was eluted off the beads twice with 100 µl SDS elution buffer (50 mM Tris pH 8.0, 1 mM EDTA and 1% SDS) at 65ºC. ChIP eluates were pooled before reversing DNA-protein cross-links. Input (filled up to 200 µl with SDS elution buffer) and ChIP samples were supplemented with 10 µl 5 M NaCl and incubated for 6-16 h at 65ºC in a hybridization oven. Samples were treated with proteinase K and RNase to remove any proteins and RNA from the co-immunoprecipitated DNA fragments. The DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) using phase-lock gel heavy microcentrifuge tubes for phase separation and precipitated with 1/70 volume of 5 M NaCl, 2 volumes of absolute ethanol and 15 µg blue glycogen (GlycoBlue). After centrifugation, the DNA pellet was air-dried and dissolved in 11 µl elution buffer (10 mM Tris-HCl, pH 8.5). The DNA concentration was determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
DNase-probed chromatin accessibility: The assay to probe chromatin accessibility with DNase was adapted to early X. tropicalis embryos using a novel approach. Ultracentrifugation- or gel electrophoresis-mediated size selection was replaced by two rounds of solid phase reverse immobilization (SPRI) to remove high molecular weight (HMW) DNA from the informative, short DNA fragments. Wide-bore pipette tips were used for the resuspensions and the transfers of biological samples from the second homogenization step until after SPRI to avoid the shearing of HMW DNA. About 250 dejellied mid-blastula embryos (stage 8+) were collected in 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold LB-DNase buffer (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM and 0.5 mM spermidine) supplemented with 0.05% Igepal CA-630. The homogenate was left on ice for 3 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was gently resuspended in 2 ml ice-cold LB-DNase buffer (without Igepal CA-630) before centrifuging (1,000 g) again for 2 min at 4ºC. The pellet was resuspended in 600 µl of room temperature LB-DNase buffer supplemented with 6 mM CaCl2. The sample was distributed equally to two 1.5-ml microcentrifuge tubes. Approximately 0.1 U DNase I was added to one aliquot while leaving the other aliquot untreated (internal control). Both samples were incubated for 8 min at 37ºC before adding 300 µl STOP buffer (50 mM Tris pH8.0, 100 mM NaCl, 100 mM EDTA, 0.1% SDS, 80 µg RNase A, 333 nM spermine and 1 µM spermidine). The samples were incubated for 15 min at 55ºC. Next, they were digested with 200 µg proteinase K for 2 h at 55ºC. The digests were transferred to phase-lock gel heavy microcentrifuge tubes for phenol:chloroform:isoamylalcohol (25:24:1) purification. The DNA was precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol. The DNA pellets were dissolved in 27 µl elution buffer (10 mM Tris pH8.5). To remove any remaining RNA (i.e. not digested in a first round of RNase treatment), 10 µg RNase A were added to the DNA samples. 5 µl (an equivalent of 20 mid-blastula embryos) from the internal control was digested with 0.3 U DNase I for 5 min at 37ºC to generate a negative control profile (i.e. DNase-treated naked genomic DNA) for chromatin accessibility. The purification and precipitation of negative control DNA was carried out along with the chromatin digest after SPRI-mediated size selection. The DNA sample from the chromatin digest was further processed by two rounds of SPRI. 22.5 µl SPRI beads were added to 25 µl DNA sample without pipetting up and down. After 3 min, by which time HMW DNA caused beads to coalesce, the tubes were clipped into a magnetic stand for microcentrifuge tubes. After 3 min, the supernatant was transferred to a 96-well microplate. 47.5 µl elution buffer and 43 µl SPRI beads were added sequentially and mixed gently by slowly pipetting up and down. After 3 min, the plate was transferred to a magnetic stand for 96-well plates. Once the beads have settled to bottom of the well, the supernatant and the digest of naked genomic DNA (see above) were transferred to separate 1.5-ml phase-lock gel heavy microcentrifuge tubes and purified with phenol:chloroform:isoamylalcohol (25:24:1, pH7.9). The DNA fragments were precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 3 volumes of absolute ethanol. After centrifugation, the DNA pellets were dissolved in 12 µl elution buffer. The DNA concentrations were determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
Chromatin conformation capture (3C): About 500 dejellied mid-blastula embryos (stage 8+) were fixed with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 40 min at room temperature. The fixation reaction was terminated by rinsing the embryos three times with ice-cold 1% MMR. The embryos were aliquoted equally into two 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold CEB-3C (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.2% SDS) supplemented with protease inhibitors and 0.05 mM DTT (=CEB-3C*). The homogenates were kept on ice for 5 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was resuspended in 0.5 ml ice-cold CEB-3C*. The resuspensions were pooled and then equally divided to two 50-ml conical tubes and each filled up with CEB-3C to 50 ml. The embryonic extracts were incubated at 37ºC for 1 h in a hybridization oven with the tubes rotating inside a hybridization bottle. The tubes were then centrifuged (1,000 g) for 5 min at room temperature. The pellets were resuspended in 50 ml double distilled water. The tubes were centrifuged (1,000 g) agai

treatment_protocol: Injections were carried out into the zygote's animal pole: MyoD-HA mRNA (80 pg); mPouV MO (5 ng Pou5f3.2 MO, 5 ng Pou5f3.3 MO and 5 ng standard control MO); mPouV/Sox3 MOs (5 ng Pou5f3.2 MO, 5 ng Pou5f3.3 MO and 5 ng Sox3 MO); β-catenin MO (5 ng); mVegT MO (10 ng); standard control MO (5-10 ng according to the dose used for the β-catenin or mVegT loss-of-function experiment); and α-amanitin (30 pg). Embryos were treated with 100 µM SB431542 or 10 µM LDN19318 from the 8-cell stage onwards to block Nodal or BMP signaling. Control embryos were treated accordingly with DMSO, the solvent of these antagonists.

value: wild-type (out-bred Nigerian)

tag: developmental stage

value: 10+ (early gastrula)

tag: treatment

value: injection of control MO

tag: chip antibody


title: PF052-94

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B04_S4_L001_R1_001.fastq.gz
B04_S4_L002_R1_001.fastq.gz
B04_S4_L003_R1_001.fastq.gz
B04_S4_L004_R1_001.fastq.gz

tag: cell type

value: shNT CTL


title: Wnt-LOF_exp2_st10p_polyA-RNA_rep3

description: canonical Wnt (β-catenin) loss-of-function, stage 10+ (early gastrula), experiment 2, poly(A)+ RNA, biological replicate 3

value: injection of β-catenin MO (canonical Wnt inhibition)


title: RNAseq_D7P14_TOX_Over_TerminallyExhausted_2

tag: genotype/variation

tag: culture condition


title: RNAseq_D7P14_TOX_Over_ProgenitorLike_2


title: Wnt-LOF_exp2_st10p_polyA-RNA_rep2

description: canonical Wnt (β-catenin) loss-of-function, stage 10+ (early gastrula), experiment 2, poly(A)+ RNA, biological replicate 2


title: NaCl+SB rep2

city: Barcelona

first: Francesc

last: Posas

address: Carrar Baldiri Reixac 10

country: Spain

institute: Cell Signaling Group, IRB Barcelona

data_processing:
RNA-sequencing reads were mapped to the hg19 genome using VAST-TOOLS “align” program (vast-tools align  -sp Hsa -c 15 -o vast_out --IR_version 2 --expr). Replicates were merged using “vast-tools merge” (-sp Hsa) and the table provided in INCLUSION_GEO.tsv was created by “vast-tools combine” (-sp Hsa). Differential splicing analysis was also performed using VAST-TOOLS (vast-tools compare --min_dPSI 15 --min_range 5 --print_dPSI –no_plot).
Genome_build: hg19
Supplementary_files_format_and_content: Results for each pre-determined alternative splicing event analysed by VAST-TOOLS have been stored in INCLUSION_GEO.tsv via the “combine” function. Further details and explanations can be found at https://github.com/vastgroup/vast-tools#combine-output-format

molecule: total RNA

source_name: HeLa whole cell

growth_protocol: HeLa cells were maintained in Dulbecco’s modified Eagle’s Medium supplemented with 10% fetal calf serum (Sigma), 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (GibCO) and were cultured in a 5% CO2 humidified incubator at 37ºC.

extract_protocol:
Total RNA was isolated using the RNAspin Mini Kit (GE Healthcare). An additional treatment with DNAse was performed using the TURBO DNA-free Kit  (Ambion inc).    Quality assessment of Total RNA samples were analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay.
Stranded mRNA-seq libraries (TruSeq) were prepared from 0.5 ng RNA

treatment_protocol:
siRNA protocol: HeLa cells were transiently transfected with SKIIP or scrambled stealth siRNAs (20 nM) using Lipofectamine RNAiMAX (Invitrogen).
72h after siRNA transfection, cells were incubated (or not) with 100 mM NaCl for 3 hours and treated (or not) with 10 μM SB203502 (SB) for 30 minutes prior to NaCl addition.

tag: cell line

value: HeLa

tag: sirnatreatment

value: scrambled siRNA

tag: sirnatreatment time

tag: pre_treatment time

value: 30 min

value: 100 mM NaCl

value: 3 hours


title: RNAseq_D7P14_TOX_Over_ProgenitorLike_1


title: Wnt-LOF_exp2_st10p_polyA-RNA_rep1

description: canonical Wnt (β-catenin) loss-of-function, stage 10+ (early gastrula), experiment 2, poly(A)+ RNA, biological replicate 1


title: cMO_exp2_st9p_polyA-RNA_rep1

description: standard control morphant, stage 9+ (late blastula), experiment 2, poly(A)+ RNA, biological replicate 1

value: 9+ (late blastula)


title: PF052-98

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B01_S8_L001_R1_001.fastq.gz
B01_S8_L002_R1_001.fastq.gz
B01_S8_L003_R1_001.fastq.gz
B01_S8_L004_R1_001.fastq.gz

tag: re-activation

tag: reactivation hours

tag: knockdown

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run


title: NaCl+SB rep1


title: SKIIP siRNA + NaCl rep1

value: SKIIP siRNA

value: untreated


title: Wnt-LOF_exp2_st9p_polyA-RNA_rep3

description: canonical Wnt (β-catenin) loss-of-function, stage 9+ (late blastula), experiment 2, poly(A)+ RNA, biological replicate 3

tag: strain

tag: developmental stage


title: PF052-92

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D01_S2_L001_R1_001.fastq.gz
D01_S2_L002_R1_001.fastq.gz
D01_S2_L003_R1_001.fastq.gz
D01_S2_L004_R1_001.fastq.gz

value: Primary human CD4+ T cells

value: Plate bound CD3/CD28 (100ng/mL)

tag: date of seqmatic run

tag: sample name (randomized)

tag: experiment number


title: NaCl rep2

tag: treatment


title: RNAseq_D7P14_pMIG_TerminallyExhausted_3

tag: genotype/variation

value: control (empty vector)

tag: tissue

value: Spleen

tag: surface marker

tag: culture condition

value: ex vivo


title: Wnt-LOF_exp2_st9p_polyA-RNA_rep2

description: canonical Wnt (β-catenin) loss-of-function, stage 9+ (late blastula), experiment 2, poly(A)+ RNA, biological replicate 2

value: wild-type (out-bred Nigerian)

tag: chip antibody


title: PF052-91

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
C03_S14_L001_R1_001.fastq.gz
C03_S14_L002_R1_001.fastq.gz
C03_S14_L003_R1_001.fastq.gz
C03_S14_L004_R1_001.fastq.gz

tag: cell type

tag: re-activation

tag: reactivation hours


title: RNAseq_D7P14_pMIG_TerminallyExhausted_2


title: NaCl rep1

tag: cell line

tag: sirnatreatment

tag: sirnatreatment time

tag: pre_treatment time


title: no stress rep2


title: Wnt-LOF_exp2_st9p_polyA-RNA_rep1

description: canonical Wnt (β-catenin) loss-of-function, stage 9+ (late blastula), experiment 2, poly(A)+ RNA, biological replicate 1

tag: strain

tag: developmental stage


title: PF052-90

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B08_S13_L001_R1_001.fastq.gz
B08_S13_L002_R1_001.fastq.gz
B08_S13_L003_R1_001.fastq.gz
B08_S13_L004_R1_001.fastq.gz

tag: knockdown

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run

tag: sample name (randomized)


title: PF052-89

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
C07_S12_L001_R1_001.fastq.gz
C07_S12_L002_R1_001.fastq.gz
C07_S12_L003_R1_001.fastq.gz
C07_S12_L004_R1_001.fastq.gz

tag: experiment number


title: RNAseq_D7P14_pMIG_TerminallyExhausted_1

tag: genotype/variation

tag: tissue

tag: surface marker

tag: culture condition


title: Wnt-LOF_exp2_st8p_polyA-RNA_rep3

description: canonical Wnt (β-catenin) loss-of-function, stage 8+ (mid-blastula transition), experiment 2, poly(A)+ RNA, biological replicate 3

value: 8+ (mid-blastula transition)

tag: treatment

tag: chip antibody


title: no stress rep1

tag: cell line

tag: sirnatreatment

tag: sirnatreatment time

tag: pre_treatment time


title: Wnt-LOF_exp2_st8p_polyA-RNA_rep2

description: canonical Wnt (β-catenin) loss-of-function, stage 8+ (mid-blastula transition), experiment 2, poly(A)+ RNA, biological replicate 2

tag: strain

tag: developmental stage


title: shIL1R #2 d10

city: New York

first: Lena

last: Lau

state: New York

address: 550 1st Ave, MSB417

institute: NYU School of Medicine

data_processing:
Sequenced reads were trimmed for adaptor sequence, then mapped to hg19 whole genome using Tophat
Number of reads per gene was determined using HTseq
Differential gene expression analysis was determined using DESeq2
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file includes normalized counts for each sample.

source_name: IMR90 hTERT

growth_protocol: IMR90 are grown on tissue-culture treated plates in MEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin

extract_protocol:
Total RNA was extracted using the Rneasy microkit (Qiagen) according to manufacturer's instructions.
Strand-specific RNA libraries were prepared using the TruSeq RNA Library Prep Kit.

treatment_protocol: 0.5ug/ml doxycyline was added to induce knockdown. Cells were then treated with ethanol as a control or 200nM tamoxifen to activate oncogenic RasERT2.

value: IMR90 hTERT

tag: cell type

value: lung fibroblast

tag: genotype

value: IL1R KD

tag: time

value: day 10

value: shIL1R senescent cells


title: RNAseq_D7P14_pMIG_ProgenitorLike_2


title: RNAseq_D7P14_pMIG_ProgenitorLike_3


title: PF052-93

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
C06_S3_L001_R1_001.fastq.gz
C06_S3_L002_R1_001.fastq.gz
C06_S3_L003_R1_001.fastq.gz
C06_S3_L004_R1_001.fastq.gz

tag: re-activation

tag: reactivation hours

tag: knockdown

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run

tag: sample name (randomized)


title: Wnt-LOF_exp2_st8p_polyA-RNA_rep1

description: canonical Wnt (β-catenin) loss-of-function, stage 8+ (mid-blastula transition), experiment 2, poly(A)+ RNA, biological replicate 1

tag: treatment

value: injection of β-catenin MO (canonical Wnt inhibition)

tag: chip antibody


title: PF052-87

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D02_S10_L001_R1_001.fastq.gz
D02_S10_L002_R1_001.fastq.gz
D02_S10_L003_R1_001.fastq.gz
D02_S10_L004_R1_001.fastq.gz

value: shmTOR

tag: experiment number


title: RNAseq_D7P14_pMIG_ProgenitorLike_1

tag: strain

tag: genotype/variation

tag: tissue

tag: surface marker

tag: infection status

value: chronic

tag: culture condition


title: PF052-88

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D07_S11_L001_R1_001.fastq.gz
D07_S11_L002_R1_001.fastq.gz
D07_S11_L003_R1_001.fastq.gz
D07_S11_L004_R1_001.fastq.gz

tag: cell type

tag: re-activation

tag: reactivation hours


title: Chipseq_Input_P14_Cl13_D7_ProgenitorLike_2

description: sample 20

library_source: genomic

data_processing:
alignment: Raw sequencing data were processed with CASAVA 1.8.2 to generate FastQ files. ChIP-seq reads were mapped to the mouse genome (build mm10) with Bowtie 1.1.1.
peaks: Peaks were identified using MACS (v 1.4.3; default p-value threshold of 1E-5) .
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files for  histone modification ChIP-seq

molecule: genomic DNA

extract_protocol:
cell isolation protocol: Cells from spleen were obtained by mechanical disruption. Isolated cells were further sorted based on their surface markers.
Lymphocytic choriomeningitis virus infection protocol: For acute viral infections, mice were intravenously (i.v.) injected with 2×10^5 plaque-forming unit (PFU) LCMV Armstrong. For chronic viral infections, mice were intravenously (i.v.) injected with 2×10^6 PFU LCMV clone 13.
One million FACS sorted cells were used for ChIP. DNA-protein crosslinking, nuclei isolation, and chromatin sonication were performed using truChIP Chromatin Shearing Kit (Covaris) according to manufacturer’s instructions. ChIPmentation was performed according to the published protocol (Schmidl, C., Rendeiro, A. F., Sheffield, N. C., & Bock, C. (2015). ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nature Methods, 12(10), 963–965. http://doi.org/10.1038/nmeth.3542).
The purified libraries were multiplexed and captured on an Illumina flow cell for cluster generation. Libraries were sequenced for 50 single read cycles on HiSeq 3000  according to the manufacturer's protocols.


title: shScr #2 d10

tag: cell line

tag: genotype

value: WT

tag: time

tag: developmental stage

value: control senescent cells


title: shIL1R #1 d10


title: PF052-86

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B06_S9_L001_R1_001.fastq.gz
B06_S9_L002_R1_001.fastq.gz
B06_S9_L003_R1_001.fastq.gz
B06_S9_L004_R1_001.fastq.gz

value: Primary human CD4+ T cells

tag: knockdown

value: shNT CTL

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run

tag: sample name (randomized)


title: PF052-85

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
C01_S8_L001_R1_001.fastq.gz
C01_S8_L002_R1_001.fastq.gz
C01_S8_L003_R1_001.fastq.gz
C01_S8_L004_R1_001.fastq.gz

value: Plate bound CD3/CD28 (100ng/mL)

tag: experiment number


title: DNase_st8_mPS-LOF_rep1

description:
DNase, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 1
dnase_mPSkd_st8p_exp12_xt7_unique.bw

tag: strain

tag: treatment

value: injection of mPouV/Sox3 MOs

tag: chip antibody


title: DNase_naked-gDNA_rep1

description:
DNase, stage 8+ (mid-blastula transition), naked genomic DNA, biological replicate 1; made six libraries from same DNA, sequenced independently
naked_gDNA_dnase_xt7_unique.bw


title: PF052-84

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D04_S7_L001_R1_001.fastq.gz
D04_S7_L002_R1_001.fastq.gz
D04_S7_L003_R1_001.fastq.gz
D04_S7_L004_R1_001.fastq.gz

tag: cell type

tag: re-activation


title: DNase_naked-gDNA_rep2

description:
DNase, stage 8+ (mid-blastula transition), naked genomic DNA, biological replicate 2; made two libraries from same DNA, sequenced  independently
naked_gDNA_dnase_xt7_unique.bw


title: DNase_st8_ctrl_rep1

description:
DNase, stage 8+ (mid-blastula transition), control, biological replicate 1
dhs_st8p_xt7.bed
dnase_ctrl_st8p_exp12_xt7_unique.bw


title: shIL1R #2 d4

tag: cell line

value: day 4

value: shIL1R cells beginning to senesce


title: Chipseq_Input_P14_Cl13_D7_TerminallyExhaused_1

description: sample 17

tag: tissue

value: Spleen

tag: surface marker

tag: infection status

tag: culture condition

value: ex vivo


title: Chipseq_Input_P14_Arm_D7_MemoryPrecursor_2

description: sample 16

value: acute


title: PF052-83

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B07_S6_L001_R1_001.fastq.gz
B07_S6_L002_R1_001.fastq.gz
B07_S6_L003_R1_001.fastq.gz
B07_S6_L004_R1_001.fastq.gz

tag: reactivation hours

tag: knockdown

tag: drug


title: input_st20_bud_ChIP

description:
ChIP input, stage 20 (early tailbud), embryonic bud only
input_xt7_unique.bw

source_name: embryonic bud

value: wild-type (out-bred Nigerian)

tag: developmental stage

value: 20 (early tailbud stage)


title: shScr #1 d4

value: IMR90 hTERT

value: lung fibroblast

tag: genotype

tag: time

value: control cells beginning to senesce


title: PF052-81

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
D05_S4_L001_R1_001.fastq.gz
D05_S4_L002_R1_001.fastq.gz
D05_S4_L003_R1_001.fastq.gz
D05_S4_L004_R1_001.fastq.gz

value: shmTOR

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run

tag: sample name (randomized)

tag: experiment number


title: shIL1R #2 d0

tag: cell line

tag: cell type

value: IL1R KD

value: day 0

value: shIL1R growing cells


title: shIL1R #1 d4


title: input_st20_trunk_ChIP

description:
ChIP input, stage 20 (early tailbud), embryonic trunk only
input_xt7_unique.bw

source_name: embryonic trunk

tag: strain

tag: treatment

tag: chip antibody


title: shIL1R #1 d0


title: Chipseq_Input_P14_Arm_D7_MemoryPrecursor_1

description: sample 15

tag: tissue

tag: surface marker

tag: infection status

tag: culture condition


title: shScr #2 d4

tag: genotype

value: WT

tag: developmental stage


title: input_st20_head_ChIP

description:
ChIP input, stage 20 (early tailbud), embryonic head only
input_xt7_unique.bw

source_name: embryonic head


title: Chipseq_H3K27Ac_P14_Cl13_D7_ProgenitorLike_2

description: sample 12

value: chronic

value: abcam ab4729


title: shScr #1 d10

tag: time

value: day 10

value: control senescent cells


title: Chipseq_Input_P14_Cl13_D7_TerminallyExhaused_2

description: sample 18

value: Spleen


title: Chipseq_Input_P14_Cl13_D7_StemLike_1

description: sample 19

value: ex vivo


title: shScr #1 d0

value: control growing cells


title: shScr #2 d0


title: Chipseq_H3K27Ac_P14_Cl13_D7_ProgenitorLike_1

description: sample 11


title: hDE_ScrgRNA_H3K27me3_rep2

city: Worcester

first: Rene

last: Maehr

state: MA

address: 55 Lake Ave N

department: Program in Molecular Medicine

institute: UMass Medical School

description: (no peak calls used)

data_processing:
Base calling: fastq files were obtained from Illumina Basespace under default parameters.
Bulk RNA-seq data utilized in atacTFAP analysis were trimmed, aligned, quantified, filtered, and tested for differential expression as follows.
Trimming: raw reads were trimmed 5bp from the head orientation and to 55 bp at the tail.
Alignment: reads were aligned with salmon version 0.8.2 to human reference transcriptome version GRCH37 using default parameters and setting the flags -l A --posBias --gcBias --seqBias.
Quantification: Transcript and gene expression levels were quantified using the salmon output and the txImport R package.
Filtering: we removed all genes with less than 3 (20th percentile) or more than 27337.16 (99.5th  percetnile) reads across the dataset.
Differential expression analysis:  performed using DESeq2. Genes with a minimal expression level of 5 RPKM in at least 2 samples and a FDR threshold of 0.01 were considered as differentially expressed.
ATAC-seq and ChIP-seq data underwent alignment, read extension, peak calling, IDR-based filtering, and differential analysis as follows.
Extension: For ChIP-seq only, reads were extended to 200 bp.
Alignment: reads were aligned to the human reference genome hg19 using Bowtie 2 version 2.3.2 using default parameters and filtering duplicate reads.
Peak calling and IDR: MACS2 in combination with the IDR framework was used for peak calling and detection of regions of genomic enrichment using an IDR cutoff of 0.1.
Differential analysis: we used the R package diffbind in combination with DESeq2 for all IDR based peak sets, requiring no overlap of peaks across conditions and using a DBA score based quantification.
Note: paired-end: R1 is a barcode
For RNA-seq, ATAC-seq, and ChIP-seq data visualization, IGV tools {Thorvaldsdottir:2013iw} was used to generate .tdf files.
Genome_build: hg19
Supplementary_files_format_and_content: Processed files are bed files containing peaks.  Also included is one Excel file with total RNA results from hESC and hEND. Peaks were not used for K27me3 data, so no bed files were used in the analysis.

source_name: Definitive endoderm

growth_protocol: H1-AAVS1-TetOn-dCas9-KRAB hESCs were maintained in mTeSR1 on hES qualified matrigel-coated plates

extract_protocol:
ATAC-seq samples were processed according to Buenrostro et. al with slight modifications. Briefly, approximately 50,000 cells were pelleted at 750 x g for 15 minutes at 4oC. After the addition of lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAl CA-630), nuclei were pelleted at 750 x g for 15 minutes at 4oC and the supernatent was discarded. Transposase reaction was performed at 37oC for 30 minutes. The purified, tagmented DNA was amplified for 9-10 cycles and size selected using AMPure XP beads (Beckman Coulter, A63881) as follows: 0.55X volume of 2X concentrated AMPure beads was added to the amplified DNA and incubated for 5 minutes at room temperature. The supernatant, containing low-molecular weight DNA, was collected in a separate tube by removing the beads using a magnetic rack. 1X volume of AMPure beads was added to the supernatant and incubated for 5 min at room temperature. The beads were washed twice with 75% ethanol and then air dried. The final purified library was eluted in EB buffer and sequenced (paired-end, 75-75) on a NextSeq500 or Hi-Seq2000 platform.
Total RNA was isolated using Trizol Reagent (Invitrogen, 15596-018) according to the manufacturer’s instructions. For quantitative PCR analysis, 1 μg of total RNA was reverse-transcribed using SuperScript III First-Strand Synthesis System (Thermo, 18080051).
Bulk RNA-sequencing library preparation of H1 hESC and differentiated hEND was performed following a published protocol {Zhang:2012kc}. Briefly, 4 μg of total RNA was depleted of ribosomal RNA using Ribo-Zero rRNA Removal Kit (Epicentre, MRZH116) and then converted to cDNA. Final sequencing libraries were generated using dUTP (Thermo, R0133) incorporation and uracil-N-glycosylase (NEB, M0280) treatment to generate strand-specific RNA-seq libraries. The final libraries were sequenced at paired-end on a Hi-Seq2000 platform.

treatment_protocol: For perturbation experiments, H1-AAVS1-TetOn-dCas9-KRAB hESCs were transduced with individual gRNA lentiviral constructs and selected with puromycin for 72 hours. 48 hours prior to the start of differentiation, the selected cells were pooled and treated with 500 ng/mL doxycycline. The cells were then split for differentiation and differentiated along published protocols in the presence of 500 ng/mL doxycycline.

tag: assay

tag: treatment protocol

value: Scramble gRNA

value: Millipore, 07-452


title: input_st12_ChIP_rep1

description:
ChIP input, stage 12+ (late gastrula), biological replicate 1
input_xt7_unique.bw

value: wild-type (out-bred Nigerian)

value: 12+ (late gastrula)


title: hDE_FOXA2gRNA_H3K27me3_rep2

description: (no peak calls used)

value: FOXA2 gRNA

value: Millipore, 07-450


title: input_st11_ChIP

description:
ChIP input, stage 11 (mid-gastrula)
input_xt7_unique.bw

value: 11 (mid-gastrula stage)


title: PF052-82

description:
Matched RNAseq, untargeted metabolomics, and O-link (from supernatants)
processed data file:
Experiment6_PlateBound_Counts.csv
Processed data file column headers with technical replicate IDs:
B02_S5_L001_R1_001.fastq.gz
B02_S5_L002_R1_001.fastq.gz
B02_S5_L003_R1_001.fastq.gz
B02_S5_L004_R1_001.fastq.gz

value: Primary human CD4+ T cells

tag: re-activation

value: Plate bound CD3/CD28 (100ng/mL)

tag: reactivation hours

tag: knockdown

value: shNT CTL

tag: drug

tag: donor

tag: plate # (submitted to seqmatic)

tag: seqmatic run

tag: date of seqmatic run


title: hDE_ScrgRNA_H3K27me3_rep1

description: (no peak calls used)

tag: chip antibody

value: Millipore, 07-451


title: Chipseq_Input_P14_Arm_D7_ShortLivedEffector_2

description: sample 14

tag: strain

tag: tissue

tag: surface marker

value: acute


title: Chipseq_Input_P14_Arm_D7_ShortLivedEffector_1

description: sample 13

tag: infection status

tag: culture condition


title: hDE_FOXA2gRNA_H3K27me3_rep1

description: (no peak calls used)

value: Millipore, 07-449


title: input_st13_ChIP

description:
ChIP input, stage 13+ (early neurula)
input_xt7_unique.bw

tag: developmental stage

value: 13+ (early neurula)

tag: treatment


title: Chipseq_H3K27Ac_P14_Cl13_D7_TerminallyExhaused_1

description: sample 9

value: Spleen

value: chronic

value: abcam ab4729


title: input_st10p_ChIP_rep1

description:
ChIP input, stage 10+ (early gastrula), biological replicate 1
input_xt7_unique.bw

value: 10+ (early gastrula)


title: uninj_exp5_st10p_polyA-RNA_rep2

description: uninjected, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 2


title: input_st9p_ChIP

description:
ChIP input, stage 9+ (late blastula)
input_xt7_unique.bw

value: 9+ (late blastula)


title: uninj_exp5_st10p_polyA-RNA_rep3

description: uninjected, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 3

value: wild-type (out-bred Nigerian)


title: Chipseq_H3K27Ac_P14_Arm_D7_MemoryPrecursor_2

description: sample 8

value: ex vivo


title: Chipseq_H3K27Ac_P14_Arm_D7_ShortLivedEffector_2

description: sample 6


title: Chipseq_H3K27Ac_P14_Arm_D7_MemoryPrecursor_1

description: sample 7


title: uninj_exp5_st9p_polyA-RNA_rep3

description: uninjected, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 3


title: uninj_exp5_st10p_polyA-RNA_rep1

description: uninjected, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 1


title: input_st8p_ChIP_rep2

description:
ChIP input, stage 8+ (mid-blastula transition), biological replicate 2
input_xt7_unique.bw

value: 8+ (mid-blastula transition)


title: input_st8p_ChIP_rep1

description:
ChIP input, stage 8+ (mid-blastula transition), biological replicate 1
input_xt7_unique.bw


title: Chipseq_H3K27Ac_P14_Cl13_D7_TerminallyExhaused_2

description: sample 10


title: Chipseq_H3K27Ac_P14_Arm_D7_ShortLivedEffector_1

description: sample 5


title: uninj_exp5_st9p_polyA-RNA_rep2

description: uninjected, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 2


title: input_st8_ChIP_rep2

description:
ChIP input, stage 8 (1,000-cell), biological replicate 2; sequenced twice
input_xt7_unique.bw

value: 8 (1,000-cell stage)


title: input_st10p_ChIP_rep3

description:
ChIP input, stage 10+ (early gastrula), biological replicate 3
input_xt7_unique.bw


title: uninj_exp5_st8p_polyA-RNA_rep3

description: uninjected, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 3


title: input_st7_ChIP

description:
ChIP input, stage 7 (128-cell)
input_xt7_unique.bw

value: 7 (128-cell stage)


title: uninj_exp5_st9p_polyA-RNA_rep1

description: uninjected, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 1


title: H1_END_Scr_gRNA_H3K27ac_ChIPseq_rep2

tag: assay

tag: treatment protocol

value: Scramble gRNA

value: Diagenode, C15410196


title: input_st6_ChIP

description:
ChIP input, stage 6 (32-cell)
input_xt7_unique.bw

value: 6 (32-cell stage)


title: uninj_exp5_st8p_polyA-RNA_rep2

description: uninjected, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 2


title: input_st10p_ChIP_rep2

description:
ChIP input, stage 10+ (early gastrula), biological replicate 2
input_xt7_unique.bw


title: H3K4me1_st8p_mPS-LOF_ChIP_rep2

description:
H3K4me1 ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 2
h3k4me1_mPSkd_st8p_xt7_exp12_unique.bw

value: injection of mPouV/Sox3 MOs

value: rabbit polyclonal anti-H3K4me1 (ab8895, Abcam)


title: H1_END_Scr_gRNA_H3K27ac_ChIPseq_rep1


title: uninj_exp5_st8p_polyA-RNA_rep1

description: uninjected, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 1


title: H3K4me1_st8p_mPS-LOF_ChIP_rep1

description:
H3K4me1 ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 1
h3k4me1_mPSkd_st8p_xt7_exp12_unique.bw


title: cMO_exp5_st10p_polyA-RNA_rep3

description: standard control morphant, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 3

value: injection of control MO


title: cMO_exp5_st10p_polyA-RNA_rep2

description: standard control morphant, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 2


title: 20K-midlog_R6

type: RNA

city: La Tronche

first: Thomas

last: Hindré

address: Campus de la Merci

country: France

institute: Université Grenoble Alpes-CNRS

hyb_protocol: The biotinylated cDNA spiked with bioB, bioC, bioD and cre (Hybridization Control) was hybridized to an Affymetrix E. coli Genome 2.0 Microarray for 16 hours at 45ºC. All arrays were washed and stained in an Affymetrix GeneChip Fluidics Station.

scan_protocol: Stained arrays were scanned with an Affymetrix GeneChip® Scanner 3000.

data_processing: Quality check and data analysis were carried out using Affymetrix GeneChip Command Console (AGCC) and Expression Console.

label: biotin

organism: Escherichia coli B str. REL606

source_name: REL8593A isolated from population Ara-1 at 20,000 generations

label_protocol: Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 10 ug total RNA.

growth_protocol: Bacteria were acclimated during 24h, 37°C, 120rpm in DM25, then diluted 100 times in 200mL DM25 and grown until mid-exponential phase.

extract_protocol: RNA was extracted using Qiagen RNeasy Cell Tissue Kit, following the manufacturer's protocol.

treatment_protocol: Bacterial cells were pelleted at 4°C.

value: REL606 derivative

tag: population

tag: generation


title: H3K4me1_st8p_ctrl_ChIP_rep2

description:
H3K4me1 ChIP, stage 8+ (mid-blastula transition), control, biological replicate 2
h3k4me1_ctrl_st8p_xt7_exp12_unique.bw


title: cMO_exp5_st10p_polyA-RNA_rep1

description: standard control morphant, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 1


title: H1_END_Scr_gRNA_ATACseq_rep1

value: ATAC


title: H1_END_Scr_gRNA_ATACseq_rep2


title: H3K4me1_st8p_ctrl_ChIP_rep1

description:
H3K4me1 ChIP, stage 8+ (mid-blastula transition), control, biological replicate 1
h3k4me1_ctrl_st8p_xt7_exp12_unique.bw


title: cMO_exp5_st9p_polyA-RNA_rep3

description: standard control morphant, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 3


title: RNAPII_st8p_mPS-LOF_ChIP_rep2

description:
RNAPII ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 2
pol2_mPSkd_st8p_xt7_exp12_unique.bw

value: mouse monoclonal anti-RNAPII (MMS-126R, Covance)


title: cMO_exp5_st9p_polyA-RNA_rep2

description: standard control morphant, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 2


title: RNAPII_st8p_mPS-LOF_ChIP_rep1

description:
RNAPII ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 1
pol2_mPSkd_st8p_xt7_exp12_unique.bw


title: cMO_exp5_st9p_polyA-RNA_rep1

description: standard control morphant, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 1


title: 20K-midlog_R3


title: RNAPII_st8p_ctrl_ChIP_rep2

description:
RNAPII ChIP, stage 8+ (mid-blastula transition), control, biological replicate 2
pol2_ctrl_st8p_xt7_exp12_unique.bw


title: H1_END_FOXA2_TFChIPseq_rep2

value: TF ChIP

value: No guide

value: Millipore, 07-633


title: H1_END_FOXA2_TFChIPseq_rep1


title: 20K-midlog_R4


title: cMO_exp5_st8p_polyA-RNA_rep3

description: standard control morphant, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 3

value: wild-type (out-bred Nigerian)


title: 20K-midlog_R2


title: cMO_exp5_st8p_polyA-RNA_rep2

description: standard control morphant, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 2


title: H1_END_FOXA2_gRNA_H3K27ac_ChIPseq_rep2

value: FOXA2 gRNA


title: 20K-midlog_R1


title: H1_END_FOXA2_gRNA_ATACseq_rep2


title: H1_END_FOXA2_gRNA_H3K27ac_ChIPseq_rep1


title: 20K-midlog_R5


title: 2K-midlog_R6

source_name: REL1164A isolated from population Ara-1 at 2,000 generations


title: cMO_exp5_st8p_polyA-RNA_rep1

description: standard control morphant, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 1

value: 8+ (mid-blastula transition)


title: 2K-midlog_R5


title: Smad2_st8p_mPS-LOF_ChIP_rep1

description:
Smad2 ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 1
smad2_mPSkd_st8p_xt7_exp12_unique.bw

value: goat polyclonal anti-Smad2/3 (AF3797, R&D Systems)


title: 2K-midlog_R3


title: Smad2_st8p_ctrl_ChIP_rep2

description:
Smad2 ChIP, stage 8+ (mid-blastula transition), control, biological replicate 2
smad2_ctrl_st8p_xt7_exp12_unique.bw


title: mVegT-LOF_exp5_st10p_polyA-RNA_rep3

description: mVegT loss-of-function, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 3

value: 10+ (early gastrula)

value: injection of mVegT MO


title: 2K-midlog_R2


title: Smad2_st8p_mPS-LOF_ChIP_rep2

description:
Smad2 ChIP, stage 8+ (mid-blastula transition), mPouV/Sox3 loss-of-function, biological replicate 2
smad2_mPSkd_st8p_xt7_exp12_unique.bw


title: H1_END_FOXA2_gRNA_ATACseq_rep1


title: RNAPII_st8p_ctrl_ChIP_rep1

description:
RNAPII ChIP, stage 8+ (mid-blastula transition), control, biological replicate 1
pol2_ctrl_st8p_xt7_exp12_unique.bw


title: 2K-midlog_R4


title: mVegT-LOF_exp5_st10p_polyA-RNA_rep2

description: mVegT loss-of-function, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 2


title: mVegT-LOF_exp5_st9p_polyA-RNA_rep3

description: mVegT loss-of-function, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 3

value: 9+ (late blastula)


title: Smad2_st8p_ctrl_ChIP_rep1

description:
Smad2 ChIP, stage 8+ (mid-blastula transition), control, biological replicate 1
smad2_ctrl_st8p_xt7_exp12_unique.bw


title: mVegT-LOF_exp5_st10p_polyA-RNA_rep1

description: mVegT loss-of-function, stage 10+ (early gastrula), experiment 5, poly(A)+ RNA, biological replicate 1


title: RNAPII_st10p_MyoD-HA_ChIP_rep1

description:
RNAPII ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 1
pol2_st10p_oe_myod_xt7_exp12_unique.bw
pol2_st10p_oe_myod_xt7.bed

value: Injection of MyoD-HA mRNA


title: mVegT-LOF_exp5_st9p_polyA-RNA_rep2

description: mVegT loss-of-function, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 2


title: r1021Lm1

city: Madrid

first: José Luis

last: Trejo

state: Madrid

address: Avenida Doctor Arce 37

institute: Instituto Cajal

description:
TREJO_01_C_SED_B_vs_C_RUN_B_without_outlier_results.xls
TREJO_01_without_outlier_norm_counts_CRIAS

data_processing:
RNA-Seq reads were mapped against the Mus musculus reference genome (GRCm38 gencode version M14) with STAR version 2.5.2a (ENCODE parameters for long RNA).
Genes were quantified with RSEM version 1.2.28 (with default parameters).
Normalization and differential expression were performed with DESeq2 version 1.10.
Genome_build: GRCm38 gencode version M14
Supplementary_files_format_and_content: All processed data are .xls files. "TREJO_01_PADRES_SED_B_vs_PADRES_RUN_B_without_outlier_results.xls" contains baseMean, log2fold change, lfcMLE, lfcSE, stat, pvalue and padj values for each gene in the progenitors comparison. Fold change represents gene expression level of exercised fathers with respect to the sedentary ones. "TREJO_01_C_SED_B_vs_C_RUN_B_without_outlier_results.xls" contains baseMean, log2fold change, lfcMLE, lfcSE, stat, pvalue and padj values for each gene in litters comparison. Fold change represents gene expression level of litters from exercised fathers with respect to the litters from sedentary fathers. "TREJO_01_Sample_Stats" contains reading values for every sample. "TREJO_01_without_outlier_norm_counts_CRIAS" and "TREJO_01_without_outlier_norm_counts_PADRES" contains counts for every gene and every sample from litters and fathers respectively.

source_name: Hippocampus

extract_protocol:
The QuickGene RNA tissue Kit SII (RT-S2) and the QG-Mini80 (Kurabo) was used to extract total RNA from hippocampal tissue. Extraction was performed following the manufacturer’s instructions with slight modifications. As the total amount of tissue per sample was lower than 15 milligrams, the final volume of the lysate buffers was doubled. Two columns of the QG-Mini80 equipment were used per sample. After treating the tissue with DNase (Promega), a third and a fourth wash with the wash buffer was added to the protocol to improve RNA purity. After letting the membrane of the column dry completely in order to remove possible traces of ethanol, samples were eluted in 22 microlitres of CRT buffer to gain a greater sample concentration. Next, the concentration of total RNA was measured by Nanodrop 1000.
The quality control for quantity and quality of the total RNA was done using the Qubit® RNA HS Assay (Life Technologies) and RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent). The experimental protocol to construct the RNASeq libraries starting from the total RNA employed the TruSeq®Stranded mRNA LT Sample Prep Kit (Illumina Inc., Rev.E, October 2013). Briefly, 500ng of total RNA was used as the input material and was enriched for the mRNA fraction using oligo-dT magnetic beads. The mRNA was fragmented in the presence of divalent metal cations and at high temperature (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, this allowed to achieve the strand specificity. The blunt-ended double-stranded cDNA was 3´adenylated and Illumina indexed adapters were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay (Agilent). The libraries were sequenced on HiSeq2000 (Illumina, Inc) in a paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4. In a mean, we generated 56 million paired-end reads for each sample in a fraction of a sequencing v4 flow cell lane, following the manufacturer’s protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.18.66.3) and followed by generation of FASTQ sequence files by CASAVA.

tag: Sex

value: Male

tag: genotype

value: wild type

tag: age

value: 5.5 months

tag: group

value: litters from exercised fathers

value: born from an exercised father

tag: sample barcode


title: mVegT-LOF_exp5_st9p_polyA-RNA_rep1

description: mVegT loss-of-function, stage 9+ (late blastula), experiment 5, poly(A)+ RNA, biological replicate 1

tag: chip antibody


title: Sox3_st10p_MyoD-HA_ChIP_rep2

description:
Sox3 ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 2
sox3_st10p_oe_myod_xt7_exp12_unique.bw
sox3_st10p_oe_myod_xt7.bed

tag: strain

value: rabbit polyclonal anti-Sox3 (Klymkowsky Lab)


title: mVegT-LOF_exp5_st8p_polyA-RNA_rep3

description: mVegT loss-of-function, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 3


title: Day3_Rep1 quantseq

description:
polyadenylated RNA
Day3_Rep1_Scr_gRNA1
quantseq_timecourse.quant.gene.txt

data_processing:
ScreenProcessing (https://github.com/mhorlbeck/ScreenProcessing) was used to quantify guide abundance in genomic DNA.
QuantSeq reads were aligned using hisat2 v2.0.5 with hg19 as a reference and parameters  “-p 12 --rna-strandness F”. Quantification used ESAT v0.1 with parameters “-wLen 100 -wOlap 50 -wExt 1000 -sigTest .01 -multimap normal”
10X Genomics Cellranger version 2.1.0 was used for fastq generation, alignment, and quantification of 10X data (definitive endoderm). The reference FASTA and GTF were prepended with customized sequences and annotations (attached as supplemental files) to facilitate alignment and tagging of guide RNAs and dCas9-KRAB.
Drop-seq tools version 1.0 was used for alignment and quantification of drop-seq data (hepatic endoderm).
Alignment and quantification were performed using the STAR aligner v2.4.2, Picard tools v1.96, Samtools v1.3, and Drop-seq tools v1.0. Details follow.
Note: fastq (R1 is a barcode)
Analysis step: Convert to SAM  Software package:  Picard tools   Command name:  FastqToSam.jar Parameters used:  --
Analysis step: Tag cell barcode  Software package:  DropSeq tools  Command name: TagBamWithReadSequenceExtended  Parameters used:  BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1,  DISCARD_READ=False
Analysis step: Tag molecular barcode  Software package:  DropSeq tools  Command name: TagBamWithReadSequenceExtended Parameters used:  BASE_QUALITY=10, BARCODED_READ=1, NUM_BASES_BELOW_QUALITY=1,  DISCARD_READ=True
Analysis step: Remove low-quality barcodes  Software package:  DropSeq tools  Command name:   FilterBAM  Parameters used:     --
Analysis step: Remove polyA tail  Software package:  DropSeq tools  Command name:   PolyATrimmer               Parameters used:  MISMATCHES=0, NUM_BASES=6
Analysis step: Convert to FASTQ  Software package:  Picard tools   Command name:   SamToFastq.jar             Parameters used:     --
Analysis step: Align reads to exome  Software package:  STAR  Command name:   --  Parameters used: --
Analysis step: Sort BAM to speed merging  Software package:  Picard tools  Command name:   SortSam.jar                 Parameters used:     --
Analysis step: Merge barcode and alignment info  Software package:  Picard tools  Command name:   MergeBamAlignment.jar       Parameters used:  INCLUDE_SECONDARY_ALIGNMENTS=False
Analysis step: Label read with exon  Software package:  DropSeq tools  Command name:   TagReadWithGeneExon        Parameters used:     --
Analysis step: Screen for bead errors  Software package:  DropSeq tools  Command name:  DetectBeadSynthesisErrors  Parameters used: NUM_BARCODES=2000
Analysis step: Form DGE matrix  Software package:  DropSeq tools  Command name:   DigitalExpression          Parameters used: MIN_NUM_GENES_PER_CELL=1000
Genome_build: hg19 plus guide RNA and dCas9-KRAB sequences
Supplementary_files_format_and_content: Processed files are digital gene expression matrices containing UMI counts. For Drop-seq data (hepatic endoderm), they are represented as dense matrices in (gzipped) tab-delimited text files. Each column is labeled with a cell barcode and each row is labeled with a gene. For 10X data (definitive endoderm), they are represented as gzipped TAR archives containing MEX-format digital gene expression matrices.
Supplementary_files_format_and_content: DE_TERA_rep1.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep2.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep1_gAmp.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: DE_TERA_rep2_gAmp.tar.gz: Gzipped tar archive of MEX format digital gene expression matrix
Supplementary_files_format_and_content: star_perturbseq_HE_rep1_merged_gene_exon_tagged_clean.dge.txt: Dense digital gene expression matrix (gzipped tab-separated text)
Supplementary_files_format_and_content: star_perturbseq_HE_rep2_merged_gene_exon_tagged_clean.dge.txt: Dense digital gene expression matrix (gzipped tab-separated text)
Supplementary_files_format_and_content: DE1_gDNA_ryan_guides_2018JAN19_unique_only.fa.counts: Whitespace-separated two-column text file; guide names followed by counts.
Supplementary_files_format_and_content: DE2_gDNA_ryan_guides_2018JAN19_unique_only.fa.counts: Whitespace-separated two-column text file; guide names followed by counts.
Supplementary_files_format_and_content: quantseq_timecourse.quant.gene.txt: Typical ESAT output: whitespace-separated text file with header containing sample names and body containing counts. First three columns contain gene symbol, chromosome, and strand.

extract_protocol: Quant-seq Protocol (Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, 015.96)


title: r37Lm2


title: RNAPII_st10p_MyoD-HA_ChIP_rep2

description:
RNAPII ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 2
pol2_st10p_oe_myod_xt7_exp12_unique.bw
pol2_st10p_oe_myod_xt7.bed

value: mouse monoclonal anti-RNAPII (MMS-126R, Covance)


title: Sox3_st10p_MyoD-HA_ChIP_rep1

description:
Sox3 ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 1
sox3_st10p_oe_myod_xt7_exp12_unique.bw
sox3_st10p_oe_myod_xt7.bed


title: r1124Im3


title: Anc-midlog_R5

source_name: REL606 ancestral clone

value: Ancestor


title: Day1_Rep2 quantseq

description:
polyadenylated RNA
Day1_Rep2_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: Day2_Rep1 quantseq

description:
polyadenylated RNA
Day2_Rep1_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: mVegT-LOF_exp5_st8p_polyA-RNA_rep2

description: mVegT loss-of-function, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 2


title: HA_st10p_MyoD-HA_ChIP_rep2

description:
HA ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 2
myod-ha_st10p_oe_myod_xt7_exp12_unique.bw
myod-ha_st10p_oe_myod_xt7.bed

value: rabbit polyclonal anti-HA (ab9110, Abcam)


title: r37Lm1


title: Anc-midlog_R6


title: Anc-midlog_R4


title: Day1_Rep1 quantseq

description:
polyadenylated RNA
Day1_Rep1_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: Day2_Rep2 quantseq

description:
polyadenylated RNA
Day2_Rep2_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: mVegT-LOF_exp5_st8p_polyA-RNA_rep1

description: mVegT loss-of-function, stage 8+ (mid-blastula transition), experiment 5, poly(A)+ RNA, biological replicate 1

value: wild-type (out-bred Nigerian)


title: Day3_Rep2 quantseq

description:
polyadenylated RNA
Day3_Rep2_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: VegT_st10p_ChIP

description: VegT ChIP, stage 10+ (1,000-cell)

value: rabbit polyclonal anti-VegT (Gurdon Lab)


title: Anc-midlog_R2


title: 2K-midlog_R1

value: REL606 derivative


title: uninj_exp4_st10p_polyA-RNA_rep3

description: uninjected, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 3


title: HA_st10p_MyoD-HA_ChIP_rep1

description:
HA ChIP, stage 10+ (early gastrula), ectopic expression of MyoD-HA, biological replicate 1
myod-ha_st10p_oe_myod_xt7_exp12_unique.bw
myod-ha_st10p_oe_myod_xt7.bed


title: Anc-midlog_R3


title: VegT_st8_ChIP

description: VegT ChIP, stage 8 (1,000-cell)

value: 8 (1,000-cell stage)


title: Day0_Rep2 quantseq

description:
polyadenylated RNA
Day0_Rep2_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: Anc-midlog_R1


title: r1124Im2

description:
TREJO_01_C_SED_B_vs_C_RUN_B_without_outlier_results.xls
TREJO_01_without_outlier_norm_counts_CRIAS


title: Sox3_st20_bud_ChIP

description: Sox3 ChIP, stage 20 (early tailbud), embryonic bud only

value: 20 (early tailbud stage)


title: Day0_Rep1 quantseq

description:
polyadenylated RNA
Day0_Rep1_Scr_gRNA1
quantseq_timecourse.quant.gene.txt


title: uninj_exp4_st10p_polyA-RNA_rep2

description: uninjected, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 2

value: 10+ (early gastrula)


title: uninj_exp4_st9p_polyA-RNA_rep3

description: uninjected, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 3

value: 9+ (late blastula)


title: DE_TERA_gDNA_rep2

description: genomic DNA

extract_protocol: As in Datlinger, P., Rendeiro, A. F., Schmidl, C., Krausgruber, T., Traxler, P., Klughammer, J., ... & Bock, C. (2017). Pooled CRISPR screening with single-cell transcriptome readout. Nature methods, 14(3), 297.


title: DE_TERA_gDNA_rep1

description: genomic DNA


title: Sox3_st20_trunk_ChIP

description: Sox3 ChIP, stage 20 (early tailbud), embryonic trunk only


title: Sox3_st20_head_ChIP

description: Sox3 ChIP, stage 20 (early tailbud), embryonic head only


title: uninj_exp4_st10p_polyA-RNA_rep1

description: uninjected, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 1


title: HE_cropseq_rep1

description: polyadenylated RNA

source_name: Hepatic endoderm

extract_protocol: Drop-Seq Laboratory Protocol v3.1, Macosko et al. Cell 2015 (hepatic endoderm)


title: Smad2_st10p_ChIP

description: Smad2 ChIP, stage 10+ (early gastrula); sequenced twice

value: goat polyclonal anti-Smad2/3 (AF3797, R&D Systems)


title: r26Gm1

value: Male


title: uninj_exp4_st8p_polyA-RNA_rep3

description: uninjected, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 3

value: 8+ (mid-blastula transition)


title: HE_cropseq_rep2

description: polyadenylated RNA


title: s817Cm4

value: wild type

value: 5.5 months

value: litters from sedentary fathers

value: born from a sedentary father


title: s511Bm2


title: s512Am4


title: s613Fm5


title: DE_TERA_cropseq_rep2_gAmp

description: PCR-amplified guide RNAs

extract_protocol: 10x Genomics Chromium Single Cell 3' Reagent Kit v2 (definitive endoderm)


title: uninj_exp4_st9p_polyA-RNA_rep2

description: uninjected, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 2


title: uninj_exp4_st9p_polyA-RNA_rep1

description: uninjected, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 1


title: Smad1_st10p_ChIP_rep2

description:
Smad1 ChIP, stage 10+ (early gastrula), biological replicate 2
smad1_st10p_xt7_exp12_unique.bw
smad1_st10p_xt7.bed

value: rabbit polyclonal anti-phospho-Smad1/5/9 (#9511, Cell Signalling)


title: DE_TERA_cropseq_rep1_gAmp

description: PCR-amplified guide RNAs


title: Sox3_st10p_ChIP

description: Sox3 ChIP, stage 10+ (early gastrula)

value: rabbit polyclonal anti-Sox3 (Klymkowsky Lab)


title: s715Hm2


title: r26Gm4

value: litters from exercised fathers

value: born from an exercised father


title: Smad1_st10p_ChIP_rep1

description:
Smad1 ChIP, stage 10+ (early gastrula), biological replicate 1; sequenced twice
smad1_st10p_xt7_exp12_unique.bw
smad1_st10p_xt7.bed

value: wild-type (out-bred Nigerian)


title: s1327Em1


title: uninj_exp4_st8p_polyA-RNA_rep1

description: uninjected, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 1


title: mPouV-Sox3-LOF_exp4_st10p_polyA-RNA_rep3

description: mPouV/Sox3 loss-of-function loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 3

value: injection of mPouV/Sox3 MOs


title: DE_TERA_cropseq_rep2

description: polyadenylated RNA


title: uninj_exp4_st8p_polyA-RNA_rep2

description: uninjected, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 2


title: Smad2_st8_ChIP

description: Smad2 ChIP, stage 8 (1,000-cell)

value: 8 (1,000-cell stage)


title: s151Jm1

description:
TREJO_01_C_SED_B_vs_C_RUN_B_without_outlier_results.xls
TREJO_01_without_outlier_norm_counts_CRIAS


title: s818Jm1


title: O1392

city: Dublin

first: Andrew

middle: E

last: Hogan

address: OLCH Crumlin

country: Ireland

institute: National Children's Research Centre

data_processing:
Next generation sequencing was carried out using Illumina NextSeq 500 Single-End 75bp (SE75) chemistry
Single-end RNA-Seq reads were aligned to the human hg38 reference genome using Tophat2 and Bowtie and the Ensembl gene annotation downloaded from the Biomart Site
The Qlucore Omics Explorer 3.2 software package was used for visualization of RNA-Seq data and to carry out Gene Set Enrichment Analysis (GSEA)
Qlucore Omics software converted imported bam files into read counts and then normalized these across the samples using the TMM (trimmed mean of M-values) method
Read counts were converted to LogCPM values using EdgeR which were normalized across samples using the TMM (trimmed mean of M-values) method
Genome_build: NCBI Human HG38 reference genome
Supplementary_files_format_and_content: Raw read counts and normailised read counts in log CPM (counts per million). Significantly enriched pathways with CPM for each gene and sample in pathways.

source_name: morbidly obese donor_Peripheral MAIT cells

growth_protocol: MAIT cells were isolated from fresh venous blood samples using high purity FACS sorted

extract_protocol:
RNA was extracted from cells using the RNeasy Mini Isolation Kit (Qiagen) and analysed for quality control and quantification purposes using the Agilent Bioanalyzer and RNA Pico assay (Agilent Technologies)
RNA-Seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories, Inc.)

treatment_protocol: No treatment

tag: subject bmi

tag: cell type

value: Peripheral MAIT cells


title: β-catenin_st10p_ChIP

description: β-catenin ChIP, stage 10+ (early gastrula)

value: 10+ (early gastrula)

value: rabbit polyclonal anti-β-catenin (H-102, Santa-Cruz)


title: mPouV-Sox3-LOF_exp4_st10p_polyA-RNA_rep2

description: mPouV/Sox3 loss-of-function loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 2


title: DE_TERA_cropseq_rep1

description: polyadenylated RNA


title: mPouV-Sox3-LOF_exp4_st10p_polyA-RNA_rep1

description: mPouV/Sox3 loss-of-function loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 1


title: Smad1_st8p_ChIP

description: Smad1 ChIP, stage 8+ (mid-blastula transition)


title: O1384


title: O1391


title: β-catenin_st6_ChIP

description: β-catenin ChIP, stage 6 (32-cell); sequenced twice

value: 6 (32-cell stage)


title: r33

description:
TREJO_01_PADRES_SED_B_vs_PADRES_RUN_B_without_outlier_results.xls
TREJO_01_without_outlier_norm_counts_PADRES

value: Male

value: 7 months

value: exercised fathers

value: 40 minutes of treadmill exercise every day for 6 weeks


title: r22

value: wild type


title: mPouV-Sox3-LOF_exp4_st9p_polyA-RNA_rep3

description: mPouV/Sox3 loss-of-function loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 3

value: 9+ (late blastula)


title: β-catenin_st8_ChIP

description: β-catenin ChIP, stage 8 (1,000-cell); sequenced twice


title: r44


title: mPouV-Sox3-LOF_exp4_st9p_polyA-RNA_rep2

description: mPouV/Sox3 loss-of-function loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 2


title: O1383


title: mPouV-Sox3-LOF_exp4_st9p_polyA-RNA_rep1

description: mPouV/Sox3 loss-of-function loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 1


title: NS

source_name: healthy bodyweight donor_Peripheral MAIT cells


title: r111


title: RNAPII_st12p_ChIP

description: RNAPII ChIP, stage 12+ (late gastrula); sequenced twice

value: 12+ (late gastrula)

value: mouse monoclonal anti-RNAPII (MMS-126R, Covance)


title: mPouV-Sox3-LOF_exp4_st8p_polyA-RNA_rep3

description: mPouV/Sox3 loss-of-function loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 3

value: 8+ (mid-blastula transition)


title: EB


title: RNAPII_st11_ChIP

description: RNAPII ChIP, stage 11 (mid-gastrula)

value: 11 (mid-gastrula stage)


title: s133

value: sedentary fathers

value: animal without access to exercise


title: mPouV-Sox3-LOF_exp4_st8p_polyA-RNA_rep2

description: mPouV/Sox3 loss-of-function loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 2

value: wild-type (out-bred Nigerian)

value: injection of mPouV/Sox3 MOs


title: CB

value: Peripheral MAIT cells


title: RNAPII_st8p_ChIP

description: RNAPII ChIP, stage 8+ (mid-blastula transition)


title: s11


title: RNAPII_st13p_ChIP

description: RNAPII ChIP, stage 13+ (early neurula)

value: 13+ (early neurula)


title: s83


title: mPouV-Sox3-LOF_exp4_st8p_polyA-RNA_rep1

description: mPouV/Sox3 loss-of-function loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 1


title: RNAPII_st8_ChIP

description: RNAPII ChIP, stage 8 (1,000-cell); sequenced twice

value: 8 (1,000-cell stage)


title: AH


title: RNAPII_st6_ChIP

description: RNAPII ChIP, stage 6 (32-cell); sequenced twice

value: 6 (32-cell stage)


title: AOB


title: mPouV-LOF_exp4_st10p_polyA-RNA_rep3

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 3

value: 10+ (early gastrula)

value: injection of mPouV MOs


title: RNAPII_st7_ChIP

description: RNAPII ChIP, stage 7 (128-cell); sequenced twice

value: 7 (128-cell stage)


title: Haploid genetic screen  for resistance to ZIKV_PF13 infection

city: Stanford

first: Ryan

middle: Alexander

last: Flynn

state: CA

address: 380 Roth Way, Room 265

institute: Stanford University

description:
ZIKV_PF13
Lentiviral inserted genomic DNA

data_processing:
Library strategy: LAM-PCR retrieval of flanking genomic sites
Sequencing reads were collapsed for PCR duplicates by removal of identical sequences and mapped to the human (hg18) genome using bowtie
Unique reads were assigned to genes, categorized into disrupting mutations (sense intron, sense exon, antisense exon and non-disrupting mutations (antisense intron) and counted
Enrichment of the inactivating mutations in the selected dataset versus all the mutations in control datset was calculated according to Fischer exact test corrected for FDR PMID:21866103
Genome_build: hg38
Supplementary_files_format_and_content: excel file summarizing the number of insertions and enrichment p-values

source_name: Human near-haploid cell line HAP1

growth_protocol: HAP1 cells were grown in IMDM with 10% FBS and 1% penicillin-streptomycin.

extract_protocol:
DNA was isolated using the QIAamp DNA mini kit (Qiagen)
Libraries were prepared according to the LAM-PCR protocol (PMID:21866103)

treatment_protocol: Cells were mutagenized using a retroviral gene trap library as described in PMID:21866103 and exposed to Zika virus

tag: cell line

tag: gender

value: male


title: mPouV-LOF_exp4_st10p_polyA-RNA_rep2

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 2

tag: developmental stage


title: s72

value: Male

value: wild type

value: 7 months

tag: treatment


title: mPouV-LOF_exp4_st9p_polyA-RNA_rep3

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 3

value: 9+ (late blastula)


title: H2-HS: wild type HSF1

city: Houston

first: Kevin

middle: A

last: Morano

state: TX

address: 6431 Fannin St, MSB 1.300

department: Microbiology and Molecular Genetics

institute: UT Medical School at Houston

data_processing:
The original raw data from Illumina are transformed to Sequenced Reads by base calling. Raw data are recorded in a FASTQ file, which contains sequence information (reads) and corresponding sequencing quality information.
Raw reads are filtered to remove reads containing adapters or reads of low quality, so that downstream analyses are based on clean reads.
Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v.2.0.12.
HTSeq  v0.6.1 was used to count the read numbers mapped to each gene, and FPKM calculated based on gene length and mapped reads using the union mode.
Genome_build: Saccharomyces_cerevisiae.Ensembl_release81.exon.gtf
Supplementary_files_format_and_content: Tab-delimited spreadsheet with FPKM for each sample

organism: Saccharomyces cerevisiae

source_name: yeast, wild type HSF1 allele

growth_protocol: Cell cultures grown to mid-log phase in rich YPD medium at 30˚C, harvested by centrifugation and cell pellet defanted and frozen immediately -80˚C.

extract_protocol:
Hot phenol total RNA isolation
Novogene Corp library service: After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. The mRNA is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/µl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM).

treatment_protocol: As control, but shifted to 37˚ C for 15 min prior to harvest.

value: wild type HSF1

value: heat shock 37˚ C 15 min


title: mPouV-LOF_exp4_st10p_polyA-RNA_rep1

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 10+ (early gastrula), experiment 4, poly(A)+ RNA, biological replicate 1


title: H3-HS: wild type HSF1


title: mPouV-LOF_exp4_st9p_polyA-RNA_rep2

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 2


title: CP40

city: Torun

first: CHANDRA

middle: SHEKHAR

last: PAREEK

state: Kuj. Pom.

address: ul. Wilenska 4

department: Centre for Modern Interdisciplinary Technologies

country: Poland

institute: Nicolaus Copernicus University

description:
young bull
fpkm calculated using cufflinks

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to UMD cattle genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
fpkm calculated using cufflinks
Genome_build: UMD Bos Taurus plus y-chromosome
Supplementary_files_format_and_content: tab-delimited text files with option of excel file format, include RPKM values for each Sample ...

organism: Bos taurus

source_name: liver

extract_protocol:
Total RNA isolation from bovine liver tissues of three cattle breeds were harvested using Trizol reagent. Illumina TruSeq mRNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: breed

value: Hereford

tag: tissue

value: liver


title: Control cells not exposed to virus

description:
Unselected_control
Lentiviral inserted genomic DNA


title: H1-HS: wild type HSF1


title: Haploid genetic screen  for resistance to ZIKV_FLR infection

description:
ZIKV_FLR
Lentiviral inserted genomic DNA


title: CP39


title: mPouV-LOF_exp4_st9p_polyA-RNA_rep1

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 9+ (late blastula), experiment 4, poly(A)+ RNA, biological replicate 1

source_name: whole embryo

value: wild-type (out-bred Nigerian)


title: NC3: hsf1-mNmC

source_name: yeast, mutated amino- and carboxyl-terminal sites

value: untreated


title: sos1_salt

city: Sevilla

first: Javier

last: Perez Hormaeche

state: Sevilla

address: Av. Americo Vespucio

department: IBVF

institute: CSIC

description: Each array includes 3 different specific oligonucleotides recognizing the same locus. These are used as technical replicates.

hyb_protocol: Cy3- and Cy5-labelled cRNAs were combined and hybridized to the microarray for 17h at 60°C using Agilent's gaskets G2534-60002, G2534A hybridization chambers and DNA Hybridization Oven G2545A, according to the manufacturer's instructions.

scan_protocol: Raw data were obtained using Agilent's DNA Microarray Scanner G2505B and Feature Extraction software (v10.1)

data_processing: The raw fluorescence intensity data were processed using the PolyphemusTM software, developed at Oryzon Genomics. Microarray data was normalized by the median-centering method. TIGR MIDAS software was used for data normalization.

organism: Oryza sativa

source_name: salt treated sos 1 roots

label_protocol: Total RNA (0.5 μg) amplification and labelling with Cy3 or Cy5 was carried out using the MessageAmpTM RNA amplification kit from Ambion (Applied Biosystems).

growth_protocol: Control and sos1 seedlings were grown hydroponically in Miyamoto's medium for 3 weeks in environmentally controlled conditions (12 h of light at 25°C and ca. 450 µmol quanta m-2 s-1, and 12 h darkness at 21°C; relative humidity ca. 65%).

extract_protocol: Total RNA was isolated from roots of sos1 mutant and control plants that had been grown hydroponically for 15 d using the Plant Total RNA Extraction kit (Qiagen). The quality and concentration of the RNA were analysed using the Agilent 2100 Bioanalyzer and NanoDrop™ ND-1000 (Thermo Scientific). Samples with RNA integrity number (RIN) < 6.0 were discarded.

treatment_protocol: Salt stress was imposed by transferring to fresh nutrient solution containing 75 mM NaCl for 4 days. Control plants received fresh nutrient solution without NaCl.

tag: cultivar

value: Nipponbare

value: sos1 mutant

value: roots

source_name: salt treated control roots

value: azygous control line


title: sos1_control

source_name: untreated sos1 roots

source_name: untreated control roots


title: GeCKO screen for DENV-4 using Huh7.5.1 cells

description: CRISPR/Cas9 mutated genomic DNA

data_processing:
Library strategy: Genome Scale CRISPR KO (GeCKO, V2)
Sequencing reads were analyzed using MAGeCK algorithm, taking into account multiple different sgRNAs per gene, number of sequencing reads per gene, and the enrichment of sgRNAs compared to the uninfected pooled library (PMID: 25476604)
Each screen data was individually analyzed, and combined as 4 replicates for MAGeCK analysis

source_name: Dengue virus infection

growth_protocol: Huh7.5.1 cells were grown in DMEM with 10% FBS and 1% penicillin-streptomycin.

extract_protocol:
Virus resistant cells were harvested for total genomic DNA extraction using QIAamp DNA Mini Kit (Qiagen)
Libraries were prepared as previously reported (PMID: 27383987)

treatment_protocol: Cells were mutagenized using GeCKO V2 libraries (PMID:25075903)

value: Hepatocarcinoma cell line


title: mPouV-LOF_exp4_st8p_polyA-RNA_rep3

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 3

extract_protocol:
Chromatin immunoprecipitation (ChIP): Briefly, dejellied X. tropicalis embryos were treated with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 15-45 min at room temperature to cross-link chromatin proteins to nearby genomic DNA. Duration of fixation was determined empirically and depended mainly on the developmental stage and antibody epitopes as described in Gentsch and Smith (2017). Efficient Preparation of High-Complexity ChIP-Seq Profiles from Early Xenopus Embryos. Methods in Molecular Biology, 1507, 23-42. Fixation was terminated by rinsing embryos three times with ice-cold 1% MMR. If required, post-fixation embryos were dissected to select specific anatomical regions in ice-cold 1% MMR. Fixed embryos were homogenized in CEWB1 (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS) supplemented with 0.5 mM DTT, protease inhibitors and, if using phospho-specific antibodies, phosphatase blockers (0.5 mM orthovanadate and 2.5 mM NaF). To solubilise yolk platelets and separate them from the nuclei, the homogenate was left on ice for 5 min and then centrifuged (1000 g) for 5 min at 4ºC. Homogenization and centrifugation was repeated once before resuspending the nuclei containing pellet in 1-3 ml CEWB1. Nuclear chromatin was solubilized and fragmented by isothermal focused or microtip-mediated sonication. The solution of fragmented chromatin was cleared by centrifuging (15,000 g) for 5 min at 4ºC. If required, ~1% of the cleared chromatin extract was set aside for the input sample (negative control for ChIP). ChIP-grade antibodies were used to recognize specific chromatin features and to enrich these by coupling the antibody-chromatin complex to protein G magnetic beads and extensive washing. These steps were carried out at 4ºC. The beads were washed twice in CEWB1, twice in WB2 (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.1% SDS), twice in WB3 (10 mM Tris pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Igepal CA-630 and 1% sodium deoxycholate) and once in TEN (10 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA). ChIP was eluted off the beads twice with 100 µl SDS elution buffer (50 mM Tris pH 8.0, 1 mM EDTA and 1% SDS) at 65ºC. ChIP eluates were pooled before reversing DNA-protein cross-links. Input (filled up to 200 µl with SDS elution buffer) and ChIP samples were supplemented with 10 µl 5 M NaCl and incubated for 6-16 h at 65ºC in a hybridization oven. Samples were treated with proteinase K and RNase to remove any proteins and RNA from the co-immunoprecipitated DNA fragments. The DNA was purified with phenol:chloroform:isoamyl alcohol (25:24:1, pH 7.9) using phase-lock gel heavy microcentrifuge tubes for phase separation and precipitated with 1/70 volume of 5 M NaCl, 2 volumes of absolute ethanol and 15 µg blue glycogen (GlycoBlue). After centrifugation, the DNA pellet was air-dried and dissolved in 11 µl elution buffer (10 mM Tris-HCl, pH 8.5). The DNA concentration was determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
DNase-probed chromatin accessibility: The assay to probe chromatin accessibility with DNase was adapted to early X. tropicalis embryos using a novel approach. Ultracentrifugation- or gel electrophoresis-mediated size selection was replaced by two rounds of solid phase reverse immobilization (SPRI) to remove high molecular weight (HMW) DNA from the informative, short DNA fragments. Wide-bore pipette tips were used for the resuspensions and the transfers of biological samples from the second homogenization step until after SPRI to avoid the shearing of HMW DNA. About 250 dejellied mid-blastula embryos (stage 8+) were collected in 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold LB-DNase buffer (15 mM Tris-HCl pH 8.0, 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM and 0.5 mM spermidine) supplemented with 0.05% Igepal CA-630. The homogenate was left on ice for 3 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was gently resuspended in 2 ml ice-cold LB-DNase buffer (without Igepal CA-630) before centrifuging (1,000 g) again for 2 min at 4ºC. The pellet was resuspended in 600 µl of room temperature LB-DNase buffer supplemented with 6 mM CaCl2. The sample was distributed equally to two 1.5-ml microcentrifuge tubes. Approximately 0.1 U DNase I was added to one aliquot while leaving the other aliquot untreated (internal control). Both samples were incubated for 8 min at 37ºC before adding 300 µl STOP buffer (50 mM Tris pH8.0, 100 mM NaCl, 100 mM EDTA, 0.1% SDS, 80 µg RNase A, 333 nM spermine and 1 µM spermidine). The samples were incubated for 15 min at 55ºC. Next, they were digested with 200 µg proteinase K for 2 h at 55ºC. The digests were transferred to phase-lock gel heavy microcentrifuge tubes for phenol:chloroform:isoamylalcohol (25:24:1) purification. The DNA was precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol. The DNA pellets were dissolved in 27 µl elution buffer (10 mM Tris pH8.5). To remove any remaining RNA (i.e. not digested in a first round of RNase treatment), 10 µg RNase A were added to the DNA samples. 5 µl (an equivalent of 20 mid-blastula embryos) from the internal control was digested with 0.3 U DNase I for 5 min at 37ºC to generate a negative control profile (i.e. DNase-treated naked genomic DNA) for chromatin accessibility. The purification and precipitation of negative control DNA was carried out along with the chromatin digest after SPRI-mediated size selection. The DNA sample from the chromatin digest was further processed by two rounds of SPRI. 22.5 µl SPRI beads were added to 25 µl DNA sample without pipetting up and down. After 3 min, by which time HMW DNA caused beads to coalesce, the tubes were clipped into a magnetic stand for microcentrifuge tubes. After 3 min, the supernatant was transferred to a 96-well microplate. 47.5 µl elution buffer and 43 µl SPRI beads were added sequentially and mixed gently by slowly pipetting up and down. After 3 min, the plate was transferred to a magnetic stand for 96-well plates. Once the beads have settled to bottom of the well, the supernatant and the digest of naked genomic DNA (see above) were transferred to separate 1.5-ml phase-lock gel heavy microcentrifuge tubes and purified with phenol:chloroform:isoamylalcohol (25:24:1, pH7.9). The DNA fragments were precipitated with 1/20 volume of 3 M sodium acetate (pH 5.2) and 3 volumes of absolute ethanol. After centrifugation, the DNA pellets were dissolved in 12 µl elution buffer. The DNA concentrations were determined on a fluorometer using high-sensitivity reagents for double-stranded DNA (10 pg/µl to 100 ng/µl).
Chromatin conformation capture (3C): About 500 dejellied mid-blastula embryos (stage 8+) were fixed with 1% formaldehyde (in 1% Marc’s Modified Ringer’s, MMR) for 40 min at room temperature. The fixation reaction was terminated by rinsing the embryos three times with ice-cold 1% MMR. The embryos were aliquoted equally into two 2-ml round-bottom microcentrifuge tubes and homogenized in 2 ml ice-cold CEB-3C (10 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.25% sodium deoxycholate and 0.2% SDS) supplemented with protease inhibitors and 0.05 mM DTT (=CEB-3C*). The homogenates were kept on ice for 5 min before centrifuging (1,000 g) for 2 min at 4ºC. The pellet was resuspended in 0.5 ml ice-cold CEB-3C*. The resuspensions were pooled and then equally divided to two 50-ml conical tubes and each filled up with CEB-3C to 50 ml. The embryonic extracts were incubated at 37ºC for 1 h in a hybridization oven with the tubes rotating inside a hybridization bottle. The tubes were then centrifuged (1,000 g) for 5 min at room temperature. The pellets were resuspended in 50 ml double distilled water. The tubes were centrifuged (1,000 g) agai

value: 8+ (mid-blastula transition)

value: injection of mPouV MOs


title: CP38


title: Haploid genetic screen  for resistance to ZIKV_PRV infection

description:
ZIKV_PRV
Lentiviral inserted genomic DNA

value: male


title: NC2: hsf1-mNmC


title: GeCKO screen for DENV-3 using Huh7.5.1 cells

description: CRISPR/Cas9 mutated genomic DNA


title: CP37


title: CP36


title: mPouV-LOF_exp4_st8p_polyA-RNA_rep2

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 2


title: mPouV-LOF_exp4_st8p_polyA-RNA_rep1

description: mPouV (Pou5f3.2 + Pou5f3.3) loss-of-function, stage 8+ (mid-blastula transition), experiment 4, poly(A)+ RNA, biological replicate 1


title: CP35


title: GeCKO screen for DENV-1 using Huh7.5.1 cells

description: CRISPR/Cas9 mutated genomic DNA


title: GeCKO screen for DENV-2 using Huh7.5.1 cells

description: CRISPR/Cas9 mutated genomic DNA


title: C3: hsf1-mC

source_name: yeast, mutated carboxyl-terminal Hsp70 site


title: irCLIP in HUH751-ZIKV of Vigilin bioRep 2

description:
Protein enriched RNA
irCLIP_HUH751_ZIKV_Vigilin_norm_minusStrand.bw, irCLIP_HUH751_ZIKV_Vigilin_norm_plusStrand.bw

data_processing:
Library strategy: irCLIP
Sequencing reads were trimmed of 3' adaptors, collapsed for PCR duplicates, stripped of the 5' barcode, and mapped to the human (hg38) genome using bowtie2
RT stop identificaiton, gene assignment, and peak calling was performed as in FAST-iCLIP (PMID: 25411354)
Genome_build: hg38
Supplementary_files_format_and_content: bigWig file containing unique RT stops from the high confidence intersection of biological replicates of RRBP1 and vigilin irCLIP

source_name: Zika virus infection

extract_protocol:
Lysates were sonicated RRBP1-RNA complexes or vigilin-RNA complexes were isolated with anti-RRBP1 (Bethyl A303-996A) or anti-vigilin (Bethyl A303-971A) antibody.
Libraries were prepared according to the irCLIP protocol (PMID: 27111506)

treatment_protocol: Cells were UV crosslinked at 254nm for 0.3J/cm^2 to in vivo fix RNA-RBP interactions


title: uninj_exp1_st10p_polyA-RNA_rep3

description: uninjected, stage 10+ (early gastrula), experiment 1, poly(A)+ RNA, biological replicate 3

value: 10+ (early gastrula)


title: C1: hsf1-mC


title: irCLIP in HUH751-ZIKV of Vigilin bioRep 1


title: N3: hsf1-mN

source_name: yeast, mutated amino-terminal Hsp70 site


title: uninj_exp1_st10p_polyA-RNA_rep1

description: uninjected, stage 10+ (early gastrula), experiment 1, poly(A)+ RNA, biological replicate 1


title: CP32

value: Polish-HF