title: rip_RocA003 [Sample 18]

source_name: HEK 293 T-Rex_rip_RocA003

tag: genotype/variation

value: RocA 0.03 uM


title: A431 110k rep1 (EV)

status: Public on Jun 15 2016

type: SRA

city: Montreal

first: Fabio

middle: Alexis

last: Lefebvre

state: Quebec

address: 110 Ave des Pins

department: RNA biolog

country: Canada

institute: IRCM

library_source: transcriptomic

data_processing:
Read quality was confirmed using FastQC v0.10.1 and trimming was performed with Trimmomatic when deemed necessary.
Read alignment was performed using Tophat v2.0.10 on the human GRCh37/hg19 and the Drosophila BDGP5.78/dm3 genomes, respectively.
Ribosomal RNA sequences were filtered out by first mapping the reads to FASTA files of genes annotated as rRNA in Ensembl.  Remaining reads were then re-aligned to reference genomes with Tophat.
BAM files were used for expression analyses with Cuffdiff v2.2.1 without effective length correction.
Genome_build: GRCh37/hg19 (human) and BDGP5.78/dm3 (Drosophila)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

molecule: total RNA

organism: Homo sapiens

source_name: A431 EV

growth_protocol: Human HepG2 and A431 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% of a 1:100 solution of penicillin and streptomycin (pen/strep) and 10% fetal bovine serum (depleted FBS) (Wisent). Before use, FBS was depleted of bovine EVs by ultracentrifugation (110,000 g, 18h, 4°C). Human cells were cultured at 37 °C, in an atmosphere of 5% CO2 in T-flasks of 175 cm2 and routinely detached using 0.25% trypsin upon reaching 80% confluence. Drosophila D17 and S2R+ cells were respectively maintained in Shield and Sang Insect Medium (M3) and in Schneider medium. Both media were supplemented with 1% of a 1:100 pen/strep solution and 10% depleted FBS. M3 medium additionally contained insulin (20 μg/mL). Droso¬phila cells were cultured at 25°C.

extract_protocol:
EVs and corresponding cellular pellets were resuspended in 1 mL of TRIzolTM reagent (Ambion) and processed according to the manufacturer's instructions RNA extracts were purified with the RNA Clean & ConcentratorTM-5 system (Zymo Research).  In-column DNase I (New England BioLabs) treatment, RNA washes and elution steps were performed according to the manufacturer's instructions. One additional centrifugation step was included (5 min at 16,000×g) to ensure complete removal of the washing buffer.  RNA samples were eluded in 12 μL of RNAse-free water (Wisent). Absorbance distributions were immediately quantified using a NanoDrop 2000c spectrophotometer.  RNA samples were  pure ( A260/A280 ≥ 2.0)( 2.00 ≤ A260/A230 ≤ 2.25). Aliquots of ~5 ng were submitted to capillary electrophoresis on a Bioanalyzer 2100 machine (Agilent). RNAse protection assay was performed to confirm the intraluminal topology of exRNA within EVs.  D17 and HepG2 cellular RNA extracts were used as controls. RNAse A (Qiagen) was inactivated by heat (10 min at 65 ºC) and RNA was extracted as described above.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: For EV isolation, cells were kept at low passage (P<10) and cultured for approximately 48h starting from ~10^7 cells.  EVs were isolated according to an established differential ultracentrifugation protocol (Thery, C., et al. Curr Protoc Cell Biol, 2006. ). Briefly, fresh culture supernatants (~80 mL) were cleared of floating (10 min at 400×g) and dead cells (10 min at 2000×g) using a 5810 R centrifuge (Eppendorf). Cell debris were removed (30 min at 10,000×g) and EVs were pelleted (70 min at 110,000×g) using the Sw28 and the Sw32 Ti rotor (Beckman Coulter) in an L8-70M machine (Beckmann). Preparations were extensively washed with PBS and pelleted (70 min at 110,000×g) using a RP100-AT4 rotor (Sorvall) and a RC-M100 micro ultracentrifuge (Sorvall). All steps were performed at 4 °C and EVs were immediately processed for analysis.

tag: cell line

value: A431 squamous cell carcinoma cell line

tag: cell passages

tag: rna source

value: EV


title: G1p1_B2_1_1_DND41_I2

city: Rehovot

first: Omer

last: Schwartzman

address: 234 Herzl Street

country: Israel

institute: Weizmann Institute of Science

library_source: genomic

data_processing:
The data was processed using our UMI-4C pipeline. With default parameters.
Processed reads were mapped with bowtie2 with the parameters:  --reorder --end-to-end.
Genome_build: hg19
Supplementary_files_format_and_content: The processed adj files are a 3 columns tab delimited table with headers: fragment end id1 (fend1), fragment end id2 (fend2), UMI count (count)

molecule: genomic DNA

source_name: DND41 cells

growth_protocol: Human leukemia cell lines, CMK, CMY, CHRF, and K562 were grown in RPMI (GIBCO) supplemented with 10% FBS, 1% glutamine (GIBCO), penicillin (100 U/mL), and streptomycin (100 μg/mL). Wi38 cells were grown in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1% glutamine (GIBCO), penicillin (100 U/mL), and streptomycin (100 μg/mL). (GIBCO) All cell lines were grown at 37°C and 5% CO2.

extract_protocol:
10^7 cells used to generate the primary 3C template. Cells are collected, centrifuged at 1200rpm, the supernatant is removed and are resuspended in PBS/10% FBS. Then the cells are cross-linked with formaldehyde to final concentration of 2% for 10min. The reaction is quenched by addition of glycine to final concentration of 0.125M. The cells are then centrifuged and resuspended in cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, 1 tbl Protease inhibitor). The nuclei are extracted and resuspended in 450µl UPW. For digestion, we add 60µl DpnII buffer, 15µl 10% SDS and incubate on a Thermomixer (Eppendorf) 37c, 60 minutes. 75µl of 20% TX-100 are added and the tube is incubated again 37c, 60 minutes. The chromatin is then digested with HC DpnII (NEB R0543M) in three rounds as following: 200U for 2 hours, 200U overnight and 200U for 2 hours. An aliquot is taken from the tube and run on gel to inspect digestion efficiency. The enzyme is then inactivated by incubating 65c, 20min. Next, the chromatin is ligated with 15µl of HC T4 ligase (NEB M0202M), with 10x ligase buffer to final volume of 7mL and incubated overnight at 16c. Aliquot is taken from the sample and ran on gel to inspect ligation efficiency. The chromatin is then reverse cross-linked overnight at 65c with 30ul proteinase K (Sigma-Aldrich), and then 30ul  of RNase A are added and incubated for 45 minutes, 37c. The 3C DNA is then extracted with Phenol-Chloroform resuspended in 10mM Tris-Hcl pH 8 and stored in -20c.
5-10ug DNA in 200µl Elution Buffter (EB, 10mM Tris-HCl, pH 8) of 3C template is sonicated at 4°c using Bioruptor program: 5 cycles of 30s on, 60s off. The sonicated material is inspected by running 10µl on agarose gel to assure size distribution of 450-550 bps. The sonicated DNA is subjected to end-repair reaction - 20µl 10X end-repair buffer and 10µl end-repair mix (NEB E6050L) are added to the mix and incubated at 20°c for 30 minutes. The DNA is cleaned by x2.2 AmpureXP beads (Beckman Coulter) and eluted in 76µl EB. Two nested PCR reactions are used for UMI-4C library construction. The PCR reactions are performed in 50ul volume with the following reagents: 10ul GoTaq Flexi buffer (promga M792A), 3ul MgCl 25mM (promega A3511), 0.2mM dNTPs, 2µl 10mM bait primer (0.4mM final concentration), 2µl 10mM Illumina enrichment primer 2 (0.4mM final concentration), 1ul GoTaq (promga M3008) and 200ng UMI-4C DNA template. PCR program is 2 minutes 95°c, 20 cycles of 30s 95°c, 30s 56°c, 60s 72°c, final extension of 5min 72°c. Following the PCR reaction we clean the amplified DNA with x1 SPRI beads and use 75% of the eluate to a second PCR reaction. The second PCR reaction is identical to the first. The only difference is the use of the bait primer with the Illumina dangling adapter (As shown in figure 1B). In the second PCR reaction 15-18 cycles are used instead of 20. Then, A-tailing reaction is performed with 10µl NEBbuffer2, 4µl Klenow (NEB M0212M), and 10µl 10nM dATP. Following A-tailing, the 5’ ends of the DNA are dephosphorylated with 2µl of Alkaline Phosphatase (NEB M0290S) 50°c for 60 minutes, and the DNA is cleaned with AmpureXP beads (x2) and eluted in 67µl EB. The DNA template is ligated with 5µl 15µM Illumina indexed adapters (final concentration 0.4µM), 10µl ligase mix and 80µl 10X buffer (NEB M2200). To release the non-ligated strand of the adapter, the DNA is denatured at 95°c for 2 minutes and cleaned with SPRI beads x1.

tag: cell type

value: Lymphoblastic leukemia

tag: linage

value: Hematopoietic

tag: viewpoint


title: ribo_DMSO_replicate4 [Sample 13]

city: Wako

first: Shintaro

last: Iwasaki

state: Saitama

address: S205 Bioscience Bldg. 2-1 Hirosawa

department: Cluster for Pioneering Research

country: Japan

institute: RIKEN

data_processing:
Library strategy: Ribosome Profiling
For Ribosome profiling, mRNA-Seq, and RIP-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
rRNA alignment with bowtie2 v2.0.6
Transcriptome alignment with TopHat v2.0.7
Read quantitation using custom scripts
Genome_build: hg19
Supplementary_files_format_and_content: text files contain three columns: 1. transcript name;2. CDS region (amino acid) used for read counting;3. read counts
For Bind-n-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
Depleting duplicated reads with Fast-X-toolkit
Supplementary_files_format_and_content: The fasta file was made by FASTX-Tool kit FASTA/Q Collapser, to eliminate duplicated sequence. Sequence is named with two number, a running number followed by how many  times that sequence occurred.

source_name: HEK 293 T-Rex_ribo_DMSO

extract_protocol:
Rnase I footprinting, ribosome pelleting, RNA extraction
TRI-reagent (sigma) and following Direct-zol RNA-MiniPrep (zymo research).

value: HEK 293 T-REx cell

tag: treatment

value: DMSO

tag: molecule

value: total RNA


title: rip_DMSO_replicate1 [Sample 16]

data_processing:
For Ribosome profiling, mRNA-Seq, and RIP-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
rRNA alignment with bowtie2 v2.0.6
Transcriptome alignment with TopHat v2.0.7
Read quantitation using custom scripts
Genome_build: hg19
Supplementary_files_format_and_content: text files contain three columns: 1. transcript name;2. CDS region (amino acid) used for read counting;3. read counts
For Bind-n-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
Depleting duplicated reads with Fast-X-toolkit
Supplementary_files_format_and_content: The fasta file was made by FASTX-Tool kit FASTA/Q Collapser, to eliminate duplicated sequence. Sequence is named with two number, a running number followed by how many  times that sequence occurred.

source_name: HEK 293 T-Rex_rip_DMSO

extract_protocol:
Standard mRNA-seq
TRI-reagent (sigma) and following Direct-zol RNA-MiniPrep (zymo research).

value: stabley expressing SBP-tagged eIF4A


title: ribo_DMSO_replicate3 [Sample 12]


title: ribo_PP242 [Sample 11]

source_name: HEK 293 T-Rex_ribo_PP242


title: rip_DMSO_replicate2 [Sample 17]


title: rna_RocA3 [Sample 10]

source_name: HEK 293 T-Rex_rna_RocA3

value: RocA 3 uM


title: rna_DMSO_replicate2 [Sample 7]

source_name: HEK 293 T-Rex_rna_DMSO


title: ribo_RocA3 [Sample 5]

source_name: HEK 293 T-Rex_ribo_RocA3


title: ribo_RocA003 [Sample 3]

source_name: HEK 293 T-Rex_ribo_RocA003


title: ribo_RocA03 [Sample 4]

source_name: HEK 293 T-Rex_ribo_RocA03

value: RocA 0.3 uM


title: rna_RocA003 [Sample 8]

source_name: HEK 293 T-Rex_rna_RocA003


title: HMGB2_ChIPseq_PHE

city: Los Angeles

first: Tom

last: Vondriska

state: CA

address: 650 Charles E Young Dr, BH553

country: USA

institute: UCLA

data_processing:
Basecalls performed using Genome Analyzer Pipeline
Reads were aligned to the rat reference genome (rn4) using Bowtie (0.12.7), with a maximum of two allowable mismatches in the seed region (first 28 nucleotides).
Randomized reads of the same length and number as the HMGB2 data set served as control.
MACS 1.4.1 was used for peak calling, with a p-value cutoff of 10-5.
Genome_build: rn4
Supplementary_files_format_and_content: wig files were generated using MACS 1.4.1; Scores represent the number of tags along chromosomes at a 10 bp resolution

organism: Rattus norvegicus

growth_protocol: Primary neonatal rat ventricular myocytes (NRVMs) from one day old rat pups were isolated via enzymatic digestion and plated (1X penicillin-streptomycin-glutamine/10% horse serum/5% newborn calf serum/1.68% M199 salts in DMEM) for 24 hours and transferred to DMEM media containing 0.1% insulin-transferrin-sodium selenite supplement.

extract_protocol:
NRVMs were fixed (1% formaldehyde, 10 minutes), lysed in lysis buffer (50mM HEPES pH7.5/150mM NaCl/1mM EDTA pH8/1% Triton X-100/0.1% sodium deoxycholate/0.1% SDS, protease inhibitor cocktail tablet [Roche]), sonicated to fragments of 500 bp and diluted in RIPA buffer. DNA-bound protein was immunoprecipitated using anti-HMGB2 (Abcam, ab67282) and precipitated with protein A conjugated magnetic beads (Millipore, LSKMAGA10). Beads were washed (twice in wash buffer: 0.1%SDS/1%Triton X-100/2mM EDTA pH8/150mM NaCl/20mM Tris-HCl pH8; once in 500mM NaCl in wash buffer). DNA was eluted (1%SDS/100mM NaHCO3, 15 minutes, 30°C) and phenol:chloroform purified.
Samples were ligated to sequencing adapters with Illumina Paired-End sample prep kit and sequenced on Illumina Genome Analyzer IIx using paired-end sequencing.

tag: strain background

value: Sprague Dawley

tag: age

value: 1 day old

tag: treated with

value: 10μM phenylephrine (PHE, Sigma, P-6126), 48hrs

tag: chip antibody

value: HMGB2 (abcam, ab67282)


title: rna_RocA03 [Sample 9]

source_name: HEK 293 T-Rex_rna_RocA03


title: ribo_Hipp1 [Sample 15]

source_name: HEK 293 T-Rex_ribo_Hipp1

value: Hipp 1 uM


title: ribo_Hipp001 [Sample 14]

source_name: HEK 293 T-Rex_ribo_Hipp001

value: Hipp 0.01 uM


title: rna_DMSO_replicate1 [Sample 6]


title: ribo_DMSO_replicate1 [Sample 1]


title: ribo_DMSO_replicate2 [Sample 2]


title: tir1 afb123-D rep2

city: Shanghai

first: Sheng-Bo

last: He

address: 800 Rd Dongchuan

country: China

institute: Shanghai Jiao Tong University

description: replicate 2

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  primaryTranscript dataset obtained from primaryTranscript of  Arabidopsis thaliana TAIR10 gene set  using Bowtie 2 version 2.2.2  with parameters --very-fast-local
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each sample.

organism: Arabidopsis thaliana

source_name: seedling

growth_protocol: seeds were germinated in 1/2 MS plus 1% sucrose and placed at 4 degree centigrade for 3 days and then tranferred to white light for 12 h before placed in darkness or blue light for another 4 days

extract_protocol:
RNA was extracted with RNAprep plant kit from TIANGEN, followed by DNase I (Takara) treatment. Quality control was performed with Agilent Bioanalyser. mRNA was purifed with Dynabeads® Oligo (dT)25（Life).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: ecotype

tag: genotype

value: tir1 afb1 afb2 afb3 quadruple mutant

tag: light condition

value: dark

tag: growth time

value: 4.5 days


title: cry1 cry2-BL rep2

description: replicate 2

value: cry1 cry2 double mutant

value: 15 μmol/m2/s blue light


title: tir1 afb123-D rep1

description: replicate 1


title: WT-BL rep1

description: replicate 1

value: WT


title: WT-D rep1

description: replicate 1


title: mplex2_CMK_I1

source_name: CMK cells

value: Mega-Erythroleukemia

value: Hematopoietic

value: baitG1_3_5kb bait_50kb bait_165kb ANK1


title: cry1 cry2-BL rep1

description: replicate 1


title: hbb_mplex2_B2_DND41_I2

value: Lymphoblastic leukemia

value: hbb_3HS hbb_HBB hbb_HBBP1_G1 HBB_HBE HBB_HS2 HBB_HS3 HBB_HS5 HBB_HBD


title: WT-BL rep2

description: replicate 2


title: hbb_mplex2_B1_DND41_I1


title: WT-D rep2

description: replicate 2


title: mplex2_CMK_I2


title: hbb_mplex2_B2_CMK_I2


title: hbb_mplex2_B1_K562_I1

source_name: K-562 cells


title: hbb_mplex2_B1_CMK_I1


title: HMGB2_ChIPseq_basal

value: Sprague Dawley

value: Primary neonatal rat ventricular myocytes (NRVMs)


title: G1p1_B2_1_1_Wi38_I1

source_name: Wi-38 cells

value: Fibroblasts

value: Embryonic fibroblasts


title: Hba-a1_async_CaptureC_merge

city: University Park

first: Ross

last: Hardison

state: PA

address: 303 Wartik Lab

institute: Pennsylvania State University

description:
Hba-a1 promoter anchor
828_981_975_Hba-a1

data_processing:
Library strategy: Capture-C
The raw reads were processed using published scripts (https://github.com/telenius/captureC/releases).
Custom scripts in R to normalize data by the total number of reads representing fragments ligated to the anchor region in the library.
For each time point and anchor, used custom scripts in R to combine reads representing fragments ligated to the anchor region in the library into one .bedgraph file merged across restriction digestion and/or capture replicates.
Genome_build: mm9
Supplementary_files_format_and_content: .bedgraph files containing coverage of reads (read counts of ligation products to given anchor region, per thousand total reads that represent ligation products to given anchor region).

organism: Mus musculus

growth_protocol: G1E erythroblasts were previously derived through deletion of GATA1 in mouse embryonic stem cells, followed by in vitro differentiation (Weiss, Yu, and Orkin 1997). We cultured a sub-line of G1E cells, G1E-ER4, in which GATA1-ER was retrovirally transduced (referred to in main text as “G1E GATA1-ER"), as previously described (Weiss, Yu, and Orkin 1997). We retrovirally transduced G1E-ER4 cells with the YFP-MD construct (Kadauke et al. 2012) and sorted for a pool of YFP-positive cells.

extract_protocol:
After cell fixation with 1% formaldehyde for 10min and cell sorting as described above, Capture-C was performed with a double-capture procedure (Davies et al. 2016). Chromatin was digested using DpnII. We used biotin labelled DNA oligos to pull down the target restriction fragments.
Capture-C libraries were sequenced on Nextseq500 with 2x75 bp paired end sequencing.

treatment_protocol: Except where indicated in the text as uninduced, we induced cells to mature with 100nM estradiol to activate GATA1-ER. During estradiol induction, we simultaneously treated cells with nocodazole (200ng/ml) for 7h-13h, washed once, and replated into fresh medium lacking nocodazole for varying times (40min-360min), ensuring all samples are exposed to estradiol for the same duration of 13h. We fixed cells with 1% formaldehyde, stained with DAPI, and sorted on a BD FACSAria based on YFP-MD and DAPI signal.

value: G1E ER4

value: estradiol 13h

tag: transgene

value: stably expressing YFP-MD

tag: nocodazole

value: asynchronous

tag: promoter anchor

value: Hba-a1 promoter

tag: raw file ids merged


title: Hba-a1_6h_CaptureC_merge

description:
Hba-a1 promoter anchor
826_980_974_Hba-a1

tag: cell line

value: 6h release


title: Hba-a1_0h_CaptureC_merge

description:
Hba-a1 promoter anchor
823_977_971_Hba-a1

value: 0h release


title: Hba-a1_1h_CaptureC_merge

description:
Hba-a1 promoter anchor
824_978_972_Hba-a1

value: 1h release


title: 776_AsyncUninduced_Pol2_Rep3

description:
Rep3
async_uninduced_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
523_646_776
776

data_processing:
Basecalls using bcl2fastq-1.8.4, and parameters --no-eamss --mismatches 1
Mapping to reference genome mm9 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2  --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle and peaks called using MACS with parameters --format BAM --gsize 1870000000 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Filter blacklist regions from peaks, and convert the *peaks.xls file from MACs to broadpeak format (see UCSC Genome Browser for format specs)
R scripts for further steps that generate additional processed tabular files in SupplementalTables.zip are available at the following repository: https://chsiung@bitbucket.org/arjunrajlaboratory/hsiung_mitosisreactivation
Genome_build: mm9
Supplementary_files_format_and_content: .bigwig files containing read coverage (not normalized to total library size).
Supplementary_files_format_and_content: Pooled .bigWig files across biological replicates are linked as supplementary files on the Series record.
Supplementary_files_format_and_content: SupplementalTables.zip contains two tables that include Pol II RPKM's at the 5' 2.5kb region of active genes and intergenic enhancers, as well as results of principal components analysis and other analyses as described in the accompanying publication. Information on the columns are described in the README_SupplementalTablesDescription.xls file. R scripts that generate the two tables are available at the following repository: https://chsiung@bitbucket.org/arjunrajlaboratory/hsiung_mitosisreactivation

extract_protocol: All samples, including input, were processed for library construction for Illumina sequencing  using Illumina’s TruSeq ChIP Sample Preparation Kit (Illumina cat# IP-202-  1012). In brief, DNA fragments were repaired to generate blunt ends, purified using  Agencourt AMPure XP Beads (Beckman Coulter cat# A63881), and a single “A” nucleotide  was added to each end. Double-stranded Illumina adaptors were ligated to  the fragments. Ligation products were purified using Agencourt AMPure XP Beads,  and subject to size selection using SPRIselect Beads (Beckman Coulter cat# B23318)in which both a left side size selection was performed at 0.9x volume, and a right  side size selection was performed at 0.6x volume according to manufacturer’s specifications.  Library fragments were then amplified for 16 cycles of PCR and products  were purified using Agencourt AMPure XP Beads. Constructed libraries were run on  the Agilent Bioananlyzer 2100 (Agilent Technologies) using either the DNA 7500 kit  (cat# 5067-1504) or the High Sensitivity DNA kit (cat# 5067-4626) as appropriate to  determine the average size and confirm the absence of unligated adaptors. The mean  library size is approximately 330 bp.  The ChIP-seq libraries were quantitated by qPCR using the Kapa SYBR FAST  Universal kit (Kapa Biosystems) according to the Illumina’s Sequencing Library qPCR  Quantification Guide. Libraries were multiplexed and sequenced on the Illumina HiSeq  2000 using Illumina’s kits and reagents as appropriate.  We sequenced 3 biological replicates for the 0min, 60min, 90min, 180min, and  360min time points; two biological replicates for 240min time point; and one replicate  for 40min time point.

value: no estradiol

tag: antibody

value: anti-Pol II N-20 (Santa Cruz, cat# sc899)


title: 725_AsyncUninduced_input_Rep3

description:
Rep3
725

value: input


title: Cd47_1.5h_CaptureC_merge

description:
Cd47 promoter anchor
825_979_973_Cd47

value: 1.5h release

value: Cd47 promoter


title: Cd47_0h_CaptureC_merge

description:
Cd47 promoter anchor
823_977_971_Cd47


title: 646_AsyncUninduced_Pol2_Rep2

description:
Rep2
async_uninduced_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
523_646_776
646


title: Cd47_6h_CaptureC_merge

description:
Cd47 promoter anchor
826_980_974_Cd47


title: 523_AsyncUninduced_Pol2_Rep1

description:
Rep1
async_uninduced_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
523_646_776
523


title: 775_Async_Pol2_Rep3

description:
Rep3
async_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
522_645_775
775


title: 638_AsyncUninduced_input_Rep2

description:
Rep2
638


title: 773_4h_Pol2_Rep3

description:
Rep3
4h_pool.bigWig (pooled file across Rep2, Rep3)
643_773
773

value: 4h release


title: 774_6h_Pol2_Rep3

description:
Rep3
6h_pool.bigWig (pooled file across Rep2, Rep3)
521_644_774
774


title: Hbb-b1_6h_CaptureC_merge

description:
Hbb-b1 promoter anchor
826_980_974_Hbb-b1

value: Hbb-b1 promoter


title: Cd47_1h_CaptureC_merge

description:
Cd47 promoter anchor
824_978_972_Cd47


title: Myc_0h_CaptureC_merge

description:
Myc promoter anchor
823_977_971_Myc

value: Myc promoter


title: Hbb-b1_async_CaptureC_merge

description:
Hbb-b1 promoter anchor
828_981_975_Hbb-b1


title: Hbb-b1_0h_CaptureC_merge

description:
Hbb-b1 promoter anchor
823_977_971_Hbb-b1


title: Hbb-b1_1h_CaptureC_merge

description:
Hbb-b1 promoter anchor
824_978_972_Hbb-b1


title: Hba-a1_1.5h_CaptureC_merge

description:
Hba-a1 promoter anchor
825_979_973_Hba-a1


title: Cd47_async_CaptureC_merge

description:
Cd47 promoter anchor
828_981_975_Cd47


title: 772_3h_Pol2_Rep3

description:
Rep3
3h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
520_642_772
772

value: 3h release


title: 771_1.5h_Pol2_Rep3

description:
Rep3
1h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
518_641_771
771

value: G1E ER4


title: 769_1h_Pol2_Rep3

description:
Rep3
769

value: estradiol 13h


title: 768_0h_Pol2_Rep3

description:
Rep3
0h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
517_639_768
768

value: stably expressing YFP-MD


title: Hbb-b1_1.5h_CaptureC_merge

description:
Hbb-b1 promoter anchor
825_979_973_Hbb-b1


title: Myc_6h_CaptureC_merge

description:
Myc promoter anchor
826_980_974_Myc


title: Myc_async_CaptureC_merge

description:
Myc promoter anchor
828_981_975_Myc

value: asynchronous


title: ATAC-Seq 50k mESC

city: Beijing

first: Wei

last: Xie

address: zhongguancun beidajie

institute: Tsinghua University

description: Processed data file: GSE66581_atac_50k_mESC.bedGraph

data_processing:
Library strategy: ATAC-Seq
Basecalls performed using CASAVA version 1.8
ATAC-seq reads were aligned to the mm9 genome using bowtie 2.2.2, replicates of same stage were pooled together and the coverage of reads in the genome is counted
Genome_build: mm9
Supplementary_files_format_and_content: The bedgraph files counted by the number of reads falling into 100bp bin in the genome. Bed files with MACS-called peaks are available in a tar archive on the series record.

source_name: Embryonic stem cell

growth_protocol: mESCs were cultured routinely in LIF and serum medium.

extract_protocol: ATAC-Seq libraries were prepared using ATAC-seq developed by Buenrostro et al. with slightly modification

tag: strain

tag: developmental stage

value: Embryonic stem cell


title: Kit_6h_CaptureC_merge

description:
Kit promoter anchor
826_980_974_Kit

tag: treatment

value: 6h release

value: Kit promoter


title: Myc_1h_CaptureC_merge

description:
Myc promoter anchor
824_978_972_Myc

value: 1h release


title: 723_6h_input_Rep2

description:
Rep2
723


title: ATAC-Seq 1k mESC

description: Processed data file: GSE66581_atac_1k_mESC.bedGraph


title: Myc_1.5h_CaptureC_merge

description:
Myc promoter anchor
825_979_973_Myc


title: Kit_0h_CaptureC_merge

description:
Kit promoter anchor
823_977_971_Kit

value: 0h release


title: 721_3h_input_Rep2

description:
Rep2
721


title: Kit_async_CaptureC_merge

description:
Kit promoter anchor
828_981_975_Kit


title: 963_Async_H3K27Ac

data_processing:
Basecalls using bcl2fastq-1.8.4 for HiSeq and bcl2fastq-2.0 for NextSeq and parameters --no-eamss --mismatches 1
Mapping to reference genome mm9 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle generated with MACS using parameters --nomodel --shiftsize=120--format BAM --gsize 1870000000 --tsize 36 --bw 120 --mfold 12 --wig --space 1
Peaks called with Sicer V1.1 using parameters mm9 1 200 150 0.74 600 0.01
Filter blacklist regions from peaks, and convert to broadpeak format (see UCSC Genome Browser for format specs)
Genome_build: mm9
Supplementary_files_format_and_content: .bigwig files containing read coverage (not normalized to total library size)

extract_protocol: All samples were processed for library construction for Illumina sequencing  using Illumina’s TruSeq ChIP Sample Preparation Kit (Illumina cat# IP-202-  1012). In brief, DNA fragments were repaired to generate blunt ends, purified using  Agencourt AMPure XP Beads (Beckman Coulter cat# A63881), and a single “A” nucleotide  was added to each end. Double-stranded Illumina adaptors were ligated to  the fragments. Ligation products were purified using Agencourt AMPure XP Beads,  and subject to size selection using SPRIselect Beads (Beckman Coulter cat# B23318)in which both a left side size selection was performed at 0.9x volume, and a right  side size selection was performed at 0.6x volume according to manufacturer’s specifications.  Library fragments were then amplified for 16 cycles of PCR and products  were purified using Agencourt AMPure XP Beads. Constructed libraries were run on  the Agilent Bioananlyzer 2100 (Agilent Technologies) using either the DNA 7500 kit  (cat# 5067-1504) or the High Sensitivity DNA kit (cat# 5067-4626) as appropriate to  determine the average size and confirm the absence of unligated adaptors. The mean  library size is approximately 330 bp.  The ChIP-seq libraries were quantitated by qPCR using the Kapa SYBR FAST  Universal kit (Kapa Biosystems) according to the Illumina’s Sequencing Library qPCR  Quantification Guide. Libraries were multiplexed and sequenced on the Illumina HiSeq  2000 using Illumina’s kits and reagents as appropriate.

treatment_protocol: We induced cells to mature with 100nM estradiol to activate GATA1-ER. During estradiol induction, we simultaneously treated cells with nocodazole (200ng/ml) for 7h and collected them (for nocodazole blocked samples), or washed once and replated into fresh medium lacking nocodazole for the indicated time (40min). ensuring all samples are exposed to estradiol for the same duration of 13h. We fixed cells with 1% formaldehyde, stained with DAPI, and sorted on a BD FACSAria based on YFP-MD and DAPI signal. Sorting of MPM2-positive prometaphase populations and MPM2-negative interphase populations was carried out as described in (Campbell, Hsiung, and Blobel 2014).

tag: cell sorting

value: no sorting

value: anti-H3K27Ac antibody (ActiveMotif, cat# 39685, lot# 30615010)


title: Kit_1h_CaptureC_merge

description:
Kit promoter anchor
824_978_972_Kit


title: Kit_1.5h_CaptureC_merge

description:
Kit promoter anchor
825_979_973_Kit

value: 1.5h release


title: ATAC-Seq 4-cell reciprocal rep2

description:
Processed data file: GSE66581_atac_4cell_reciprocal.bedGraph
Processed data file: GSM2108704_4cell_reciprocal_re2.bedGraph

source_name: Preimplantation embryo

extract_protocol:
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer.
ATAC-Seq libraries were prepared using ATAC-seq developed by Buenrostro et al. with slightly modification

treatment_protocol: Embryos were collected from C57BL/6N mice induced to superovulation by injections of 5 IU of pregnant mare's serum gonadotrophin (Ningbo Sansheng Pharmaceutical Co, Ltd) and 5 IU of human chorionic gonadotrophin (Ningbo Sansheng Pharmaceutical Co, Ltd) 44-48 hours apart and mated to DBA/2N males.

value: C57BL/6N x DBA/2N


title: 957_40min_H3K27Ac

value: 40min release from nocodaozle block

value: 4N, YFP-MD low


title: 720_1.5h_input_Rep2

description:
Rep2
720

value: input


title: ATAC-Seq ICM rep2

description:
Processed data file: GSE66581_atac_ICM.bedGraph
Processed data file: GSM2156963_icm_re2.bedGraph

source_name: Inner cell mass

value: ICM


title: Kit_unin_CaptureC_merge

description:
Kit promoter anchor
827_982_976_Kit

value: G1E ER4

value: no estradiol


title: 724_Async_input_Rep2

description:
Rep2
724

value: estradiol 13h

value: stably expressing YFP-MD


title: 718_40min_input_Rep2

description:
Rep2
718

value: 40min release


title: 719_1h_input_Rep2

description:
Rep2
719


title: ChIP-Seq 2-cell H3K27ac

data_processing:
Basecalls performed using CASAVA version 1.8
ChIP-seq reads were aligned to the mm9 genome using bowtie 2.2.2. Rpkm in bedgraph files were counted by the number of reads falling into 100bp bin in the genome
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph

extract_protocol:
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer.
ChIP-Seq libraries were prepared using TELP developed by PengXu et al. with slightly modification (Peng, Xu, et al. "TELP, a sensitive and versatile library construction method for next-generation sequencing." Nucleic acids research (2014): gku828.)

treatment_protocol: Embryos were collected from C57BL/6N mice induced to superovulation by injections of 5 IU of pregnant mare’s serum gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) and 5 IU of human chorionic gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) 44-48 hours apart and mated to PWK males.

value: C57BL/6N x PWK

value: anti-H3K27ac (ActivMotif, catalog# 39133, lot# 16612005)


title: ATAC-Seq 4-cell reciprocal rep1

description:
Processed data file: GSE66581_atac_4cell_reciprocal.bedGraph
Processed data file: GSM2108703_4cell_reciprocal_re1.bedGraph


title: bind_RocA3_ADP_replicate2

description: Bind-n-Seq

data_processing:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
Depleting duplicated reads with Fast-X-toolkit
fasta files, reads named with "id number" - "duplication number of the read"

organism: synthetic construct

source_name: randomized synthetic RNAs

extract_protocol: RNA extraction, preadenylated linker ligation, reverse transcription, cDNA circularization, PCR amplification

value: RocA 3 uM

tag: extracted molecule

value: randomized synthetic RNAs


title: iclip_RocA3

description: iCLIP-Seq

data_processing:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
rRNA alignment with bowtie2 v2.0.6
Transcriptome alignment with TopHat v2.0.7
Depleting duplicated reads with Fast-X-toolkit
fasta files, reads named with "id number" - "duplication number of the read"

source_name: HEK 293 T-REx cell

value: HEK 293 T-REx cell

value: total RNA

value: Partial Rnase I digestion, SBP-tag purification, RNA extraction


title: 956_0h_H3K27Ac

value: Nocodazole blocked

value: 4N, YFP-MD high

value: anti-H3K27Ac antibody (ActiveMotif, cat# 39685, lot# 30615010)


title: iclip_RocA03

description: iCLIP-Seq

value: RocA 0.3 uM


title: ATAC-Seq early 2-cell alpha amanitin treatment rep2

description:
Processed data file: GSE66581_atac_2cell_early_trx_inhibitor.bedGraph
Processed data file: GSM2108702_2cell_early_trx_inhibitor_re2.bedGraph

value: C57BL/6N x DBA/2N

value: early 2-cell


title: 717_0h_input_Rep2

description:
Rep2
717

value: G1E ER4

value: 0h release

value: input


title: iclip_RocA003

description: iCLIP-Seq

value: RocA 0.03 uM


title: RNA-Seq 8-cell KD Nr5a2 rep4

data_processing:
Basecalls performed using CASAVA version 1.8
RNA-Seq reads were aligned to the mm9 genome assembly using STAR version 2.4.0, then replicates were merged together, and transcript abundance (FPKM) were calculated based on Refseq annotation using
Genome_build: mm9
Supplementary_files_format_and_content: fpkm

molecule: polyA RNA

growth_protocol: Knockdown siRNA injected embryos were cultured in CZB medium containing glutamine at 37℃ under 5% CO2 in air.

extract_protocol:
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer.
RNA-Seq libraries were prepared using Smart-Seq2 protocol with slightly modification


title: WT_BMDMs_T0_3

city: Boston

first: Ramnik

middle: J

last: Xavier

state: MA

address: 185 Cambridge Street

institute: Massachusetts General Hospital

description:
N704_C1
Stranded 3' digital gene expression
i7index1: TCCTGAGC
Barcode: GTAGTC

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were aligned to mouse genome (mm10) using BWA-0.7.4 which uses the Burrows-Wheeler transform as described by Li H. and Durbin R. (Bioinformatics 2009, 25:1754-60.)
RSEM-based quantification using known transcripts was performed using protocol described by Li et al (BMC Bioinformatics 2011, 12:323)
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files contain matrix of UMI counts

source_name: mouse cultured BMDMs (primary cells in culture)

growth_protocol: Cell were cultured in RPMI with 10% heat-inactivated FBS with pen-strep and 20ng/ml recombinant murine MCSF

extract_protocol:
Cells were washed in 1X PBS and lysed directly in Ambion mRNA direct lysis buffer.
RNA libraries were prepared as described in http://biorxiv.org/content/early/2014/03/05/003236

treatment_protocol: BMDMs were cultured for seven days and then harvested for RNA isolation

tag: gender

value: female

tag: chimera genotype

value: chimera WT--> WT

tag: tissue

value: bone marrow chimera

tag: cell type

value: macrophage

tag: age

value: 8 weeks old


title: 722_4h_input_Rep2

description:
Rep2
722

value: estradiol 13h

value: stably expressing YFP-MD

value: 4h release


title: bind_RocA3_ADP_replicate1

description: Bind-n-Seq

value: RocA 3 uM

value: randomized synthetic RNAs


title: 645_Async_Pol2_Rep2

description:
Rep2
async_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
522_645_775
645

value: asynchronous

value: anti-Pol II N-20 (Santa Cruz, cat# sc899)


title: iclip_DMSO_replicate1

description: iCLIP-Seq

value: DMSO

value: HEK 293 T-REx cell

value: total RNA

value: Partial Rnase I digestion, SBP-tag purification, RNA extraction


title: RNA-Seq 8-cell KD Nr5a2 rep2

value: C57BL/6N x DBA/2N


title: ChIP-Seq 2-cell H3K27me3

treatment_protocol: Embryos were collected from C57BL/6N mice induced to superovulation by injections of 5 IU of pregnant mare’s serum gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) and 5 IU of human chorionic gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) 44-48 hours apart and mated to PWK males. Each set of embryos at a particular stage was flushed from the reproductive tract at defined time periods after hCG administration: 43h (late 2-cell) in Hepes-buffered CZB medium. The zona pellucida of embryos selected by cell number or morphology was gently removed by treatment of 10 IU/ml pronase (Sigma P8811) for several minutes.

value: C57BL/6N x PWK

tag: chip antibody

value: anti-H3K27me3 (Diagenode, catalog# C15410069 (pAb-069-050))


title: 709_40min_Pol2_Rep3

description:
Rep3
709

value: G1E ER4

tag: transgene

value: 40min release


title: 644_6h_Pol2_Rep2

description:
Rep2
6h_pool.bigWig (pooled file across Rep2, Rep3)
521_644_774
644

tag: nocodazole

value: 6h release


title: 643_4h_Pol2_Rep2

description:
Rep2
4h_pool.bigWig (pooled file across Rep2, Rep3)
643_773
643

value: estradiol 13h


title: 642_3h_Pol2_Rep2

description:
Rep2
3h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
520_642_772
642

value: stably expressing YFP-MD

value: 3h release


title: RNA-Seq 8-cell KD control rep4


title: 641_1.5h_Pol2_Rep2

description:
Rep2
1h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
518_641_771
641

value: 1.5h release


title: RNA-Seq 8-cell KD Nr5a2 rep1


title: bind_input

description: Bind-n-Seq

value: randomized synthetic RNAs


title: 640_1h_Pol2_Rep2

description:
Rep2
640

value: 1h release


title: RNA-Seq 8-cell KD control rep2


title: RNA-Seq early 2-cell rep2

treatment_protocol: Embryos were collected from C57BL/6N mice induced to superovulation by injections of 5 IU of pregnant mare’s serum gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) and 5 IU of human chorionic gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) 44-48 hours apart and mated to DBA/2N males.

value: early 2-cell


title: RNA-Seq 8-cell KD control rep3


title: 639_0h_Pol2_Rep2

description:
Rep2
0h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
517_639_768
639

value: 0h release

value: anti-Pol II N-20 (Santa Cruz, cat# sc899)


title: RNA-Seq 8-cell KD control rep1


title: iclip_DMSO_replicate2

description: iCLIP-Seq

value: DMSO

value: HEK 293 T-REx cell

value: total RNA


title: 637_Async_input_Rep1

description:
Rep1
637

value: asynchronous

value: input


title: RNA-Seq MII oocyte rep2

source_name: Oocyte

extract_protocol:
After removing the zona pellucida, oocytes were washed with PBS and incubated in lysis buffer.
RNA-Seq libraries were prepared using Smart-Seq2 protocol with slightly modification

treatment_protocol: Collection of MII oocytes from superovulated C57BL/6N mice was performed 20h after hCG injection.

value: MII oocyte


title: RNA-Seq 8-cell KD Nr5a2 rep3

value: C57BL/6N x DBA/2N


title: 636_6h_input_Rep1

description:
Rep1
636

value: G1E ER4

value: 6h release


title: ATAC-Seq 8-cell rep2

description:
Processed data file: GSE66581_atac_8cell.bedGraph
Processed data file: GSM1933929_8cell_re2.bedGraph


title: RNA-Seq MII oocyte rep1


title: 635_4h_input_Rep1

description:
Rep1
635

value: estradiol 13h

value: stably expressing YFP-MD

value: 4h release


title: 634_3h_input_Rep1

description:
Rep1
634

value: 3h release


title: 633_1.5h_input_Rep1

description:
Rep1
633

value: 1.5h release


title: ATAC-Seq 8-cell rep1

description:
Processed data file: GSE66581_atac_8cell.bedGraph
Processed data file: GSM1933928_8cell_re1.bedGraph


title: ATAC-Seq 4-cell rep2

description:
Processed data file: GSE66581_atac_4cell.bedGraph
Processed data file: GSM1933927_4cell_re2.bedGraph


title: ATAC-Seq ICM rep1

description:
Processed data file: GSE66581_atac_ICM.bedGraph
Processed data file: GSM1933930_icm_re1.bedGraph

value: ICM


title: RNA-Seq early 2-cell rep1

value: early 2-cell


title: 527_3h_input_Rep1

description:
Rep1
527


title: 528_6h_input_Rep1

description:
Rep1
528


title: ATAC-Seq early 2-cell alpha amanitin treatment rep1

description:
Processed data file: GSE66581_atac_2cell_early_trx_inhibitor.bedGraph
Processed data file: GSM1933923_2cell_early_trx_inhibitor_re1.bedGraph


title: ATAC-Seq 2-cell rep2

description:
Processed data file: GSE66581_atac_2cell.bedGraph
Processed data file: GSM1933925_2cell_re2.bedGraph


title: ATAC-Seq 2-cell rep1

description:
Processed data file: GSE66581_atac_2cell.bedGraph
Processed data file: GSM1933924_2cell_re1.bedGraph


title: 525_1h_input_Rep1

description:
Rep1
525

value: 1h release


title: HepG2 total cellular rep2

source_name: HepG2 total cellular

value: HepG2 hepatocellular carcinoma cell line

tag: cell passages

tag: rna source

value: total cellular RNA


title: bind_RocA03 [Sample 23]

data_processing:
Library strategy: Bind-n-Seq
For Ribosome profiling, mRNA-Seq, and RIP-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
rRNA alignment with bowtie2 v2.0.6
Transcriptome alignment with TopHat v2.0.7
Read quantitation using custom scripts
Genome_build: hg19
Supplementary_files_format_and_content: text files contain three columns: 1. transcript name;2. CDS region (amino acid) used for read counting;3. read counts
For Bind-n-Seq:
Basecalling with Illumina Casava 1.8 software
3' adapter trimming with FastX-toolkit
Depleting duplicated reads with Fast-X-toolkit
Supplementary_files_format_and_content: The fasta file was made by FASTX-Tool kit FASTA/Q Collapser, to eliminate duplicated sequence. Sequence is named with two number, a running number followed by how many  times that sequence occurred.

source_name: randomized synthetic RNAs_bind_RocA03

extract_protocol:
randomized synthetic RNAs
TRI-reagent (sigma) and following Direct-zol RNA-MiniPrep (zymo research).

value: RocA 0.3 uM

tag: molecule subtype

value: randomized synthetic RNAs

tag: molecule

value: total RNA


title: 526_1.5h_input_Rep1

description:
Rep1
526

tag: cell line

tag: antibody

value: input


title: ATAC-Seq early 2-cell rep2

description:
Processed data file: GSE66581_atac_2cell_early.bedGraph
Processed data file: GSM1933922_2cell_early_re2.bedGraph

tag: strain

value: C57BL/6N x DBA/2N

tag: developmental stage


title: bind_DMSO [Sample 21]

source_name: randomized synthetic RNAs_bind_DMSO

tag: treatment

value: DMSO


title: 521_6h_Pol2_Rep1

description:
Rep1
6h_pool.bigWig (pooled file across Rep2, Rep3)
521_644_774
521

value: G1E ER4

value: 6h release

value: anti-Pol II N-20 (Santa Cruz, cat# sc899)


title: 522_Async_Pol2_Rep1

description:
Rep1
async_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
522_645_775
522

value: estradiol 13h

value: stably expressing YFP-MD

value: asynchronous


title: ATAC-Seq early 2-cell rep1

description:
Processed data file: GSE66581_atac_2cell_early.bedGraph
Processed data file: GSM1933921_2cell_early_re1.bedGraph


title: bind_input [Sample 20]

source_name: randomized synthetic RNAs_bind_input


title: 520_3h_Pol2_Rep1

description:
Rep1
3h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
520_642_772
520

value: 3h release


title: 631_0h_input_Rep1

description:
Rep1
631

value: 0h release


title: 632_1h_input_Rep1

description:
Rep1
632

value: 1h release


title: RNA-Seq blastocyst KD Gata4 rep1

data_processing:
Basecalls performed using CASAVA version 1.8
RNA-Seq reads were aligned to the mm9 genome assembly using STAR version 2.4.0, then replicates were merged together, and transcript abundance (FPKM) were calculated based on Refseq annotation using cufflinks version 2.2.1
Genome_build: mm9
Supplementary_files_format_and_content: The wig files counted by the number of reads falling into 100bp bin in the genome. Tab-delimited text files include RPKM values for each sample

value: Blastocyst


title: 518_1h_Pol2_Rep1

description:
Rep1
1h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
518_641_771
518


title: RNA-Seq blastocyst KD Gata4 rep3


title: RNA-Seq blastocyst KD Gata4 rep2


title: RNA-Seq blastocyst KD control rep3


title: RNA-Seq blastocyst KD Gata4 rep4


title: 517_0h_Pol2_Rep1

description:
Rep1
0h_pool.bigWig (pooled file across Rep1, Rep2, Rep3)
517_639_768
517


title: 519_1.5h_Pol2_Rep1

description:
Rep1
519

value: 1.5h release


title: RNA-Seq blastocyst KD control rep2


title: 524_0h_input_Rep1

description:
Rep1
524


title: 989_NocMPM2pos_H3K27Ac

value: Nocodazole blocked

tag: cell sorting

value: MPM2 positive

value: anti-H3K27Ac antibody (ActiveMotif, cat# 39685, lot# 30615010)


title: 988_AsyncMPM2neg_H3K27Ac

value: MPM2 negative


title: RNA-Seq mESC rep2

extract_protocol: RNA-Seq libraries were prepared using Smart-Seq2 protocol with slightly modification

value: Embryonic stem cell


title: RNAseq B6J-nmf205-/-;Gcn2-/- rep3

city: Farmington

first: Jeffrey

last: Chuang

state: CT

address: 263 Farmington Avenue

department: Computational Biology

institute: The Jackson Laboratory for Genomic Medicine

data_processing:
Illumina HiSeq 2000
Adapter sequenced trimmed using FASTX toolkit (fastx_clipper -Q33 -a CTGTAGGCACCATCAAT -l 25 -c -n -v -i $fastqfile-o temp_clipped.fastq)
Sequenced  trimmed using FASTX toolkit (fastx_trimmer -Q33 -f 2 -l 100 -i temp_clipped.fastq -o temp_trimmed.fastq)
Resulting reads were mapped to a FASTA file containing all coding transcripts plus the last 18nts of the 5’UTR and the first 18nts of the 3’UTR downloaded from Ensembl Biomart
Reads were aligned to reference using Bowtie (bowtie -S -p 16 -n 1 -m 1 -l 23 --norc $fastafile temp_trimmed.fastq). Abundance measurements were determined by a script written to count the reads that fall into each gene.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RefSeq RNA ID and raw counts for the sample

source_name: Cerebellum

extract_protocol:
Cerebellum at 3 weeks of age from each genotype was isolated and immediately frozen in liquid nitrogen. Total RNA was extracted using Trizol reagent (Life Technologies)
After fragmentation of mRNA, libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Illumina)

tag: genotype

value: 3 weeks


title: bind_RocA003 [Sample 22]

source_name: randomized synthetic RNAs_bind_RocA003

value: RocA 0.03 uM

value: randomized synthetic RNAs

value: total RNA


title: RNAseq B6J-nmf205-/-;Gcn2-/- rep1


title: RNAseq B6J-nmf205-/-;Gcn2-/- rep2


title: RNA-Seq ICM rep5

treatment_protocol: Embryos were collected from C57BL/6N mice induced to superovulation by injections of 5 IU of pregnant mare’s serum gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) and 5 IU of human chorionic gonadotrophin(Ningbo Sansheng Pharmaceutical Co, Ltd) 44-48 hours apart and mated to DBA/2N males. ICM cells were generally identified by immunosurgery from the blastocysts.

value: C57BL/6N x DBA/2N

value: Inner cell mass


title: RNAseq B6J-Gcn2-/- rep3


title: RNA-Seq ICM rep1


title: RNAseq B6J-Gcn2-/- rep2


title: RNAseq B6J-nmf205-/- rep3


title: RNAseq B6J-nmf205-/- rep2


title: RNA-Seq ICM rep2


title: RNAseq B6J-Gcn2-/- rep1


title: RNA-Seq 4-cell rep2


title: RNA-Seq 8-cell rep2


title: RNAseq B6J-nmf205-/- rep1


title: RNA-Seq blastocyst KD control rep1


title: RNA-Seq 2-cell rep2


title: RNA-Seq mESC rep1


title: RNA-Seq 4-cell rep1


title: RNAseq B6J rep2

value: wild type


title: RNA-Seq 2-cell rep1


title: RNA-Seq zygote rep1

value: Zygote


title: RNAseq B6J rep3


title: RNA-Seq zygote rep2


title: sample6_ChIP6

city: Bethesda

first: Yuka

last: Kanno

state: MD

address: 10 Center Drive, Rm13C120

department: NIAMS

institute: NIH

data_processing:
Reads of 50 bases were aligned to the mouse genome build mm9 with Bowtie 0.12.8, allowing two mismatches. Uniquely mapped and non-redundant reads were retained.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph for ChIP-seq aligned files

source_name: mouse primary CD4+ T cells

growth_protocol: Protocol 2: Naïve cells from Foxp3-GDL reporter mice were activated with anti-CD3 (5μg/ml) and anti-CD28 (5μg/ml) and polarized in the presence of TGF-β (1ng/ml), IL-2 (100U/ml) and all-trans retinoic acid (2.5nM). After stimulation for 3 days, Foxp3-EGFP+ cells were sorted, stimulated with the phorbol ester PMA and ionomycin for 1.5 h and cross-linked for ChIP.

extract_protocol:
Cells were chemically cross linked by 1% formaldehyde. Cell lysates were made by appropriate sonication and protein-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated with Taq polymerase and dATP to yield a protruding 3- 'A' base for ligation of NEBNext adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with NEBNext index primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel, and quantitated by Qubit (Invitrogen).  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 2000 following the manufacturer's protocols.

value: Treg

tag: culture condition

value: 3 day in vitro culture for Treg, growth protocol 2

value: input whole cell lysate


title: sample4_ChIP4

tag: cell type

value: p300 (Santa Cruz sc-585)


title: RNAseq B6J rep1

tag: age


title: sample5_ChIP5

growth_protocol: Protocol 1: Naïve cells from IL-17-EGFP reporter mice were activated with anti-CD3 (2.5μg/ml) and anti-CD28 (1μg/ml) and polarized in the presence of TGF-β (10ng/ml), IL-6 (100ng/ml), anti-IFN-γ (10μg/ml) and anti-IL-4 (10μg/ml) to generate Th17 cells. After stimulation for 3 days, cells were allowed to rest for next 3 days, followed by reactivation for 3 days with anti-CD3 (1μg/ml) and anti-CD28 (1μg/ml) in the presence of IL-23 (20ng/ml). On day 9, IL-17-EGFP+ cells were sorted, stimulated with the phorbol ester PMA and ionomycin for 1.5 h and cross-linked for ChIP.

value: 9 day in vitro culture for Th17 cells, growth protocol1

tag: chip antibody


title: ATAC-Seq 200 mESC

description: Processed data file: GSE66581_atac_200_mESC.bedGraph


title: sample2_ChIP2

value: Blimp1 (Cell Signaling #9115)


title: ATAC-Seq 4-cell rep1

description:
Processed data file: GSE66581_atac_4cell.bedGraph
Processed data file: GSM1625847_4cell_re1.bedGraph


title: RNA-Seq 8-cell rep1


title: sample1_ChIP1


title: sample3_ChIP3


title: nrpd1nrpe1_Col_sRNA

first: Yanqiang

last: Li

address: 300 Longwood Ave.

department: Cardiology Department

institute: Boston Children's Hospital

data_processing:
For bisulfite sequencing data processing, adapter and low quality reads (q < 20) were trimmed first. Then the remaining clean reads were mapped to the Col-0 TAIR10 Arabidopsis thaliana genome by BSMAP aligner (whole genome bisulfite sequence MAPping program) allowing up to 4 mismatches (Xi and Li, 2009). Unique mapped reads were extracted to determine the cytosine methylation level as previously described (Lister et al., 2008).Only the cytosines with >4 reads in all samples were used for subsequent analyses.
Analysis of small RNA data was carried out according to a previous study (Gent et al., 2014). In brief, clean reads were aligned to TAIR10 genome assembly using bowtie2 with at most 2 mismatches (Langmead and Salzberg, 2012), and reads not mapped to tRNA and rRNA were retained. Then, we removed the small RNA reads aligned to Arabidopsis miRNA from miRBase (Kozomara and Griffiths-Jones, 2011), and the remaining reads were used for downstream analysis. Counts of 24 nt siRNAs for each locus were normalized to Reads Per Ten Million (RPTM)  by the total number of mapping reads as library size.
Genome_build: TAIR10
Supplementary_files_format_and_content: BS-seq WIG files represent methylation level (from 0 to 1) of each cytosine that are covered ≥4 times in all 8 samples.sRNA WIG files represent normalized RPTM (reads per ten million) counts of small RNAs that are uniquely mapped to the TAIR10 genome.

growth_protocol: Seeds were stratified for 7 days at 4 °C and grown on 1/2 Murashige and Skoog (MS) nutrient medium supplemented with 1.2% agar under long day conditions (16h light, 8h dark cycle at 22 °C) for two weeks before harvested.

extract_protocol:
Genomic DNA was extracted at BGI (Beijing Genomics Institute), Inc. Small RNA was extracted using the Trizol/PEG method as published (He et al 2009 Genes Dev; doi:10.1101/gad.1765209).
Libraries were prepared for sequencing according to the manufacturer's (Illumina, Inc) instructions.

tag: strain/ecotype background

value: Col

tag: genotype/variation

value: nrpd1nrpe1 double mutant

tag: tissue

value: whole seedling


title: nrpd1nrpe1_C24_sRNA


title: nrpd1nrpe1_C24XCol_sRNA


title: C24_sRNA


title: ColXC24_sRNA


title: C24XCol_sRNA


title: RNA sequencing in ERH1 deleted cells_2

first: Shiv

last: Grewal

address: NCI bldg 37 Rm 6068 9000 Rockville Pike

department: LBMB

institute: NCI

data_processing:
Alignment was done using Tophat.
FPKM values were calculated using cufflinks
Genome_build: asm294v2.28
Supplementary_files_format_and_content: Tab delimited text files, from cufflinks

organism: Schizosaccharomyces pombe

source_name: log. growing cells

growth_protocol: Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were first grown at 30˚C to OD600=~0.2 and then shifted to 37˚C for 5 hours.

extract_protocol:
RNA was extracted using the MasterPure™ Yeast RNA Purification Kit (epicentre)
ScriptSeq v2 RNA-Seq Library Preparation Kit was used according to manufacturer's recommendation. Libraries were analyzed using an Agilent 2100 BioAnalyzer (Agilent) and sequenced on the Illumina MiSeq platform.

treatment_protocol: Genomic deletion of CCR4 and ERH1.

value: ERH1 deletion


title: RNA sequencing in CCR4 deleted cells_2

value: CCR4 deletion


title: RNA sequencing in wild type cells

value: wild type cells (Smt0)


title: Col_sRNA

source_name: Col


title: RNA sequencing in CCR4 deleted cells_1


title: nrpd1nrpe1_ColXC24_sRNA


title: nrpd1nrpe1_C24XCol_DNA


title: RNA sequencing in ERH1 deleted cells_1


title: nrpd1nrpe1_Col_DNA


title: ColXC24_DNA


title: C24XCol_DNA


title: nrpd1nrpe1_C24_DNA


title: G1_165kb_B2_1_1_DND41_I1_MS

description:
The processed data file contains reads from two separate runs which were concatenated.
adj.G1_165kb_2_1_1.DND41_I1

data_processing:
The data was processed using our UMI-4C pipeline. With default parameters.
Processed reads were mapped with bowtie2 with the parameters:  --reorder --end-to-end.
Genome_build: hg19
Supplementary_files_format_and_content: The processed adj files are a 3 columns tab delimited table with headers: fragment end id1 (fend1), fragment end id2 (fend2), UMI count (count)

source_name: DND41 cells

extract_protocol:
10^7 cells used to generate the primary 3C template. Cells are collected, centrifuged at 1200rpm, the supernatant is removed and are resuspended in PBS/10% FBS. Then the cells are cross-linked with formaldehyde to final concentration of 2% for 10min. The reaction is quenched by addition of glycine to final concentration of 0.125M. The cells are then centrifuged and resuspended in cold lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, 1 tbl Protease inhibitor). The nuclei are extracted and resuspended in 450µl UPW. For digestion, we add 60µl DpnII buffer, 15µl 10% SDS and incubate on a Thermomixer (Eppendorf) 37c, 60 minutes. 75µl of 20% TX-100 are added and the tube is incubated again 37c, 60 minutes. The chromatin is then digested with HC DpnII (NEB R0543M) in three rounds as following: 200U for 2 hours, 200U overnight and 200U for 2 hours. An aliquot is taken from the tube and run on gel to inspect digestion efficiency. The enzyme is then inactivated by incubating 65c, 20min. Next, the chromatin is ligated with 15µl of HC T4 ligase (NEB M0202M), with 10x ligase buffer to final volume of 7mL and incubated overnight at 16c. Aliquot is taken from the sample and ran on gel to inspect ligation efficiency. The chromatin is then reverse cross-linked overnight at 65c with 30ul proteinase K (Sigma-Aldrich), and then 30ul  of RNase A are added and incubated for 45 minutes, 37c. The 3C DNA is then extracted with Phenol-Chloroform resuspended in 10mM Tris-Hcl pH 8 and stored in -20c.
5-10ug DNA in 200µl Elution Buffter (EB, 10mM Tris-HCl, pH 8) of 3C template is sonicated at 4°c using Bioruptor program: 5 cycles of 30s on, 60s off. The sonicated material is inspected by running 10µl on agarose gel to assure size distribution of 450-550 bps. The sonicated DNA is subjected to end-repair reaction - 20µl 10X end-repair buffer and 10µl end-repair mix (NEB E6050L) are added to the mix and incubated at 20°c for 30 minutes. The DNA is cleaned by x2.2 AmpureXP beads (Beckman Coulter) and eluted in 76µl EB. Two nested PCR reactions are used for UMI-4C library construction. The PCR reactions are performed in 50ul volume with the following reagents: 10ul GoTaq Flexi buffer (promga M792A), 3ul MgCl 25mM (promega A3511), 0.2mM dNTPs, 2µl 10mM bait primer (0.4mM final concentration), 2µl 10mM Illumina enrichment primer 2 (0.4mM final concentration), 1ul GoTaq (promga M3008) and 200ng UMI-4C DNA template. PCR program is 2 minutes 95°c, 20 cycles of 30s 95°c, 30s 56°c, 60s 72°c, final extension of 5min 72°c. Following the PCR reaction we clean the amplified DNA with x1 SPRI beads and use 75% of the eluate to a second PCR reaction. The second PCR reaction is identical to the first. The only difference is the use of the bait primer with the Illumina dangling adapter (As shown in figure 1B). In the second PCR reaction 15-18 cycles are used instead of 20. Then, A-tailing reaction is performed with 10µl NEBbuffer2, 4µl Klenow (NEB M0212M), and 10µl 10nM dATP. Following A-tailing, the 5’ ends of the DNA are dephosphorylated with 2µl of Alkaline Phosphatase (NEB M0290S) 50°c for 60 minutes, and the DNA is cleaned with AmpureXP beads (x2) and eluted in 67µl EB. The DNA template is ligated with 5µl 15µM Illumina indexed adapters (final concentration 0.4µM), 10µl ligase mix and 80µl 10X buffer (NEB M2200). To release the non-ligated strand of the adapter, the DNA is denatured at 95°c for 2 minutes and cleaned with SPRI beads x1.

value: Lymphoblastic leukemia

tag: linage

value: Hematopoietic

tag: viewpoint


title: nrpd1nrpe1_ColXC24_DNA


title: G1_165kb_B2_1_1_DND41_I2_MS

description:
The processed data file contains reads from two separate runs which were concatenated.
adj.G1_165kb_2_1_1.DND41_I2


title: S2R+ 110k rep2 (EV)

data_processing:
Read quality was confirmed using FastQC v0.10.1 and trimming was performed with Trimmomatic when deemed necessary.
Read alignment was performed using Tophat v2.0.10 on the human GRCh37/hg19 and the Drosophila BDGP5.78/dm3 genomes, respectively.
Ribosomal RNA sequences were filtered out by first mapping the reads to FASTA files of genes annotated as rRNA in Ensembl.  Remaining reads were then re-aligned to reference genomes with Tophat.
BAM files were used for expression analyses with Cuffdiff v2.2.1 without effective length correction.
Genome_build: GRCh37/hg19 (human) and BDGP5.78/dm3 (Drosophila)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

organism: Drosophila melanogaster

source_name: S2R+ EV

extract_protocol:
EVs and corresponding cellular pellets were resuspended in 1 mL of TRIzolTM reagent (Ambion) and processed according to the manufacturer's instructions RNA extracts were purified with the RNA Clean & ConcentratorTM-5 system (Zymo Research).  In-column DNase I (New England BioLabs) treatment, RNA washes and elution steps were performed according to the manufacturer's instructions. One additional centrifugation step was included (5 min at 16,000×g) to ensure complete removal of the washing buffer.  RNA samples were eluded in 12 μL of RNAse-free water (Wisent). Absorbance distributions were immediately quantified using a NanoDrop 2000c spectrophotometer.  RNA samples were  pure ( A260/A280 ≥ 2.0)( 2.00 ≤ A260/A230 ≤ 2.25). Aliquots of ~5 ng were submitted to capillary electrophoresis on a Bioanalyzer 2100 machine (Agilent). RNAse protection assay was performed to confirm the intraluminal topology of exRNA within EVs.  D17 and HepG2 cellular RNA extracts were used as controls. RNAse A (Qiagen) was inactivated by heat (10 min at 65 ºC) and RNA was extracted as described above.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: S2R+ embryonic cell line

tag: cell passages

tag: rna source

value: EV


title: G1_165kb_B2_1_1_k562_I2_MS

description:
The processed data file contains reads from two separate runs which were concatenated.
adj.G1_165kb_2_1_1.k562_I2

source_name: K-562 cells

value: Mega-Erythroleukemia


title: D17 total cellular rep2

source_name: D17 total cellular

tag: cell line

value: Dm-D17-c3 third instar larva cell line

value: total cellular RNA


title: G1_165kb_B2_1_1_CMK_I1_MS

description:
The processed data file contains reads from two separate runs which were concatenated.
adj.G1_165kb_2_1_1.CMK_I1

source_name: CMK cells


title: G1_165kb_B2_1_1_CMK_I2_MS

description:
The processed data file contains reads from two separate runs which were concatenated.
adj.G1_165kb_2_1_1.CMK_I2


title: D17 total cellular rep1


title: D17 110k rep1 (EV)

source_name: D17 EV


title: C24_DNA

value: wild type


title: D17 110k rep1(EV)


title: S_1061

city: Gothenburg

first: Adil

last: Mardinoglu

state: Gothenburg

address: Kemivägen 10

department: Department of Biology and Biological Engineering

country: Sweden

institute: Chalmers University of Technology

data_processing:
standard Illumina RNA-Seq protocol was used for processing.
processed reads were mapped to the human genome (GRCh37) using Tophat v2.0.8b (C. Trapnell et al., Bioinformatics, 2009)
fragments per kilobase of exon model per million mapped reads (FPKM) values were calculated using Cufflinks v2.1.1 (C. Trapnell et al., Nat. Biotechnol. 2010) and the gene models from Ensembl build 75 were used in Cufflinks.
Genome_build: GRCh37
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

source_name: Subcutaneous fat tissue of obese patient

extract_protocol:
Samples were obtained from obese subjects undergoing bariatric surgery.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Subcutaneous fat

tag: bmi


title: S_1100


title: S_1040


title: 4cseq_CMK

description: CMK cells processed with 4C-seq protocol (van de Werken, 2012, nature methods)

value: bait_50kb bait_165kb ANK1 G1p1


title: S_1054


title: S_1011


title: S_1045


title: Col_DNA


title: L_1100

source_name: Liver tissue of obese patient

value: Liver


title: S_1008


title: L_1104


title: L_1055


title: L_1058


title: L_1061


title: L_1054


title: L_1045


title: Jhd2-12V5 anti-V5 ChIP

city: Salt Lake City

first: Timothy

last: Parnell

state: UT

address: 2000 Circle of Hope

institute: Huntsman Cancer Institute

description: ChIP DNA

data_processing:
De-multiplexed Fastq files from the Illumina pipeline were aligned to the Saccharomyces cerevisiae genome release 64 (UCSC SacCer3) using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Alignments were converted to sorted, indexed Bam files using samtools.
Nucleosome fragment coverage was generated using the BioToolBox bam2wig application (v1.35) by extending each alignment in the 3' direction by 150 bp and counting the coverage. Depth was normalized to reads per million (RPM). Coverage tracks were converted to bigWig files for visualization and analysis using UCSC utilities.
Genome_build: SacCer3
Supplementary_files_format_and_content: BigWig files representing nucleosome fragment (adjusted to 150 bp length) coverage depth normalized to reads per million mapped.

organism: Saccharomyces cerevisiae

source_name: Mnase-digested soluble chromatin

growth_protocol: Yeast were grown in YPAD broth (1% Yeast extract, 2% Peptone, 2% Dextrose, and 0.004% adenine hemisulfate) at 30°C with constant shaking at 225 rpm to an OD A600  of 0.8-1.0.

extract_protocol:
Yeast strains were grown to mid-log phase and cross-linked with 1% formaldehyde for 20 min at room temperature. Crosslinking was stopped using 125 mM glycine. Cells were washed extensively with 1X phosphate-buffered saline (PBS). Four sets of 100 × 107 cross-linked cells per strain were harvested and stored at -80°C. Cells were thawed, converted to spheroplasts using Lyticase (250 units, Sigma) and lysed with 0.25% Triton X-100 before isolating crude nuclei using a sucrose cushion. Nuclei were washed with micrococcal nuclease (MNase) digestion buffer (25 mM Tris.Cl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2) and resuspended in the same buffer prior to digesting chromatin with 20 units of MNase (Worthington Biochemicals) for 15 min at 37°C. Digestion was stopped by adding 10X stop buffer (50 mM Tris.Cl pH 7.5, 500 mM NaCl, 100 mM EDTA, 20 mM EGTA, 10% Triton X-100) and clarified by high speed centrifugation (16,100 X g for 10 min at 4°C). Clarified supernatants for each strain obtained from four sets of cross-linked cells were pooled together (~4.5 ml). 50 μl was set aside for isolating control input DNA. The remaining soluble chromatin was split equally into four tubes (1.3 ml each) and pre-cleared using 25 μl blocked Dynabeads® Protein G for 1h at 4°C with end-over-end rotation. Blocking of magnetic beads was performed at 4°C with end-over-end rotation for >24h using a solution made up of FA buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), 140 mM NaCl, bovine serum albumin (BSA) (0.2 mg ml-1, New England Biolabs) and fish skin gelatin (0.5%, Sigma). Blocked beads were washed with binding buffer (MNase digestion buffer + 1X stop buffer) before use. 15 μl α-V5 antibody (Bio-Rad, Catalog No. MCA1360) was then added to pre-cleared chromatin and incubation was performed overnight at 4°C with end-over-end rotation. 50 μl blocked Dynabeads® Protein G was added to capture the antigen-antibody complexes and incubation was continued for an additional 2.5 h. Beads were then sequentially washed 5 min each with end-over-end rotation at 4°C as follows: three washes with FA buffer + 140 mM NaCl, two washes with FA buffer + 1000 mM NaCl, twice with FA buffer + 500 mM NaCl, and one wash each with LiCl/NP40 buffer (10 mM Tris.Cl, pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate) and TE (10 mM Tris.Cl, pH 8.0, 1 mM EDTA). ChIP DNA was eluted in 400 μl elution solution (1% SDS, 100 mM sodium bicarbonate) and incubated at room temperature with end-over-end mixing for 40 min. The eluate is transferred to a fresh 0.65 ml tube, 18 μl 5 M NaCl is added and mixed well. At this stage, 400 μl elution solution and 18 μl 5 M NaCl is also added to the 50 μl soluble chromatin set aside to isolate ‘‘input’’ DNA. Reverse crosslinking is done by incubation at 65 °C for 5 h in a thermal cycler. The solution was transferred to a fresh 1.5 ml tube, 1 ml ethanol was added, mixed well and precipitation was allowed to occur overnight at 20 °C. The samples were centrifuged at maximum speed in a table-top centrifuge at 4°C. The pellet is washed with 70% ethanol followed by centrifugation at high speed. The pellet was dissolved in 90 μl water, treated with 2 μl 10 mg/ml RNase A and followed by an incubation at 37 °C for 30 min. Proteins were removed by adding 10 μl 10X PNK buffer (100 mM Tris.Cl, pH 8.0, 50 mM EDTA, 5% SDS), 1 μl Proteinase K (Sigma) and incubating at 42°C for 1 h. DNA was purified using Qiaquick PCR Purification kit after addition of 20 μl 3 M Sodium acetate to adjust the pH. DNA was eluted in 50 μl water and stored at -20 °C. DNA concentration was measured using Qubit Fluorometer. Equal amount ChIP or input DNA was used in library construction using NEBNext® Multiplex Oligos (Index Primers Set 1) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina.
Equal amount of purified DNA  was subjected to library construction using the NEBNext®  ChIP-Seq Library Prep Reagent Set for Illumina following the manufacturer's protocol. Adaptor-ligated and index primer-containing amplified DNA  was resolved in a 2.0 % agarose gel and DNA corresponding to ~300 bp was excised and purified using the Qiagen Minelute Gel Extraction Kit, except incubation to melt the gel was performed at 37oC for 30min. The library was then subjected to 50bp single read sequencing in an Illumina Hiseq2000 sequencer.

tag: strain

value: Jhd2-12V5::KanMX6 Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: S_1055


title: L_1040


title: L_1038


title: S_1038


title: 8V5-Set1 anti-V5 ChIP

description: ChIP DNA

value: 8V5-Set1::URA3 Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: No tag anti-V5 ChIP

description: ChIP DNA

value: HTABWT

value: Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Jhd2-12V5 input

description: genomic DNA


title: L_1036


title: L_1011


title: No tag input

description: genomic DNA

value: Mat a hta1-htb1deltaelta hta2-htb2deltaelta lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: L_1008


title: jhd2D H3-2Flag ChIP repeat2

description: ChIP DNA

data_processing:
Prior to sequence alignment, de-multiplexed Fastq files from the Illumina pipeline were pre-processed using the custom script ChIPNexus_fastq_barcode_processor (https://github.com/tjparnell/HCI-Scripts), which removes the random and fixed barcode portions from the read and stores the random sequence in the read name.
Processed fastq files were aligned using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Alignments were converted to sorted, indexed Bam files using samtools.
Alignments were de-duplicated using the custom script ChIPNexus_bam_dedup (https://github.com/tjparnell/HCI-Scripts), which uses alignment position, the stored random barcode sequence in the read name, and read quality to eliminate PCR duplicate artifacts.
De-duplicated alignment start positions were recorded as normalized counts using the bam2wig program (https://github.com/tjparnell/bioboolbox) and coverted to bigWig format using UCSC wigToBigWig utilities.
Genome_build: SacCer3
Supplementary_files_format_and_content: BigWig files representing the counts of read fragment start positions normalized to reads per million mapped.

source_name: Sonicated soluble chromatin

growth_protocol: Yeast strains were grown overnight in YPAD (1% Yeast extract, 2% Peptone, 2% Dextrose, and 0.004% adenine hemisulfate) medium containing hygromycin (200 μg ml-1) at 30°C with constant shaking at 225 rpm. Overnight cultures were diluted 1:10 into fresh YPAD medium and grown for 30 h. Cells were then grown overnight in the presence of 2 μM β-estradiol (E-8875, Sigma) to induce V5 to Flag tag switch. Overnight culture for each strain was diluted into fresh YPAD medium, and cells were arrested in G1 phase by growing them for 4 h in the presence of α-factor (5 ng μl-1, Zymo Research). G1 cells from each strain were subjected to formaldehyde crosslinking.

extract_protocol:
Formaldehyde cross-linked G1 arrested yeast cells were lysed with bead beating and sonicated in a Diagenode Bioruptor™ for 30 min (6 cycles of 5 min each, 30 sec ON and 30 sec OFF at high setting). Soluble chromatin was obtained after two sequential high-speed centrifugations of the sonicated lysate (16,100 X g for 5 min and 15 min). SDS was removed by dialysis as follows. Soluble chromatin obtained from four batches of control or mutant budding yeast and candida admixture were pooled, transferred to a 5 ml Float-A-Lyzer G2 dialysis device (3.5 - 5 kD MWCO) (Spectra/Por) and dialyzed initially for 2 h in 1 L FA140 buffer followed by an overnight dialysis in fresh 4 L FA140 buffer. Dialysate was centrifuged at 16,100 X g for 15 min at 4°C. A 100 μl aliquot was set aside for isolating input DNA. The remaining dialysate was split equally into three tubes and pre-cleared using 50 μl blocked Dynabeads® Protein G for 1h at 4°C with end-over-end rotation. Dynabeads® Protein G were blocked with BSA and fish skin gelatin for 4 h. Pre-cleared chromatin was used for immunoprecipitation with 15 μl α-V5 (Bi-Rad) or 15 μl α-Flag M2 (Sigma) antibody along with the blocked Dynabeads® Protein G and incubated overnight with end-over-end rotation.
ChIP-nexus library preparation was performed following the procedure described by Zeitlinger and colleagues (He et al (2015) Nature Biotechnology 33, 395–401). Briefly, ChIP DNA on beads was washed sequentially with Buffer A (10 mM TE, 0.1% Triton X-100), Buffer B (20mM Tris.Cl pH 8.0, 150mM NaCl, 5 mM EDTA, 5.2% sucrose, 1.0% Triton X-100, 0.2% SDS), Buffer C (5 mM Tris.Cl pH 8.0, 250 mM NaCl, 25 mM HEPES.KOH pH 7.5, 0.5% Triton X-100, 0.05% sodium deoxycholate, 0.5 mM EDTA) and Buffer D (10 mM Tris.Cl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 10 mM EDTA). Bead-bound ChIP DNA was subjected to end repair, dA-tailing, Nexus adapter ligation, 5’ overhang fill-in, 5’ end trim, λ exonuclease and RecJ f exonuclease digestions. Beads were washed with Buffers A-D and equilibrated in 10 mM Tris.Cl, whose pH was same as that of the buffer used in the next enzymatic step. Finally, beads were washed 3 times with RIPA buffer (50 mM HEPES pH 7.5, 1 mM EDTA, 0.7% sodium deoxycholate, 1% NP-40, 0.5 M LiCl). ChIP DNA was extracted from beads using an elution buffer (50 mM Tris.Cl pH 8.0, 10 mM EDTA, 1% SDS) and incubation at 65°C in a Thermomixer with shaking. After addition of equal volume of TE, reversal of crosslinking was performed at 65°C for 6 h followed by RNase A and Proteinase K digestion. ChIP DNA was subjected to phenol and phenol-chloroform extractions before addition of glycogen (10 μg μl-1) and ethanol precipitation. Purified DNA was dissolved in 12 μl water, and 1μl was used in qPCR to check ChIP efficiency at the PMA1 5’ region. The remaining 11 μl ChIP DNA was denatured at 95°C for 5 min followed by single-strand DNA circularization using CircLigase (Epicentre) at 60°C for 1 h. The cut oligo was then annealed to the circularized single strand DNA in a Thermocycler (95°C for 1 min, 25°C for 1 min (1% ramp), 25°C for 30 min), linearized using FastDigest BamHI (Fisher Scientific) before ethanol precipitation. ChIP DNA libraries from control and mutant strains were PCR amplified using universal and barcoded primers present in the NEB NEBNext® Multiplex Oligos Index Primers Set 1. Library DNA was gel purified using Qiagen MinElute Gel Extraction kit.

value: jhd2delta::KanMX6; HHT1-V5-LoxP-HphMX-LoxP-2FLAG PTDH3-Cre-EBD78::URA3 (hht2-hhf2)delta::NatMX6 Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: set1_3

description:
Set1_Plus.bw
Set1_Minus.bw
set1∆  Repeat 3

data_processing:
Splice junctions based on UCSC SacCer3 gene annotation were generated using the USeq application MakeTranscriptome with a radius of 46 bp. An alignment index was created using genomic sequence, splice junction sequences, PhiX, and adaptor sequences. Fastq sequence reads were aligned against this genome index using Novoalign (v2.8.1) allowing for up to 50 hits. Raw alignments were converted to processed alignments using the USeq application SamTranscriptomeParser to convert junction alignments into genomic coordinates, discard multiple alignments, and filter for alignment scores < 90. Alignments were written as sorted indexed Bam files.
Stranded read coverage for each experimental condition was generated using the USeq application Sam2USeq. Alignments from each replicate were merged into a single file. Coverage depth was normalized as Reads Per Million mapped (RPM).
Differential gene expression was determined using the USeq application DefinedRegionDifferentialSeq (v8.8.1), which uses the DESeq R package to apply variance normalization between replicates and determine significantly different expression based on a binomial model. Gene models were based on UCSC SacCer3 annotation. Analysis was performed twice, looking first at sense transcription, then at antisense transcription.
Genome_build: SacCer3
Supplementary_files_format_and_content: RNASeq stranded coverage normalized to Reads Per Million mapped

source_name: mid-log phase yeast cells

growth_protocol: Yeast were grown in YPAD broth (1% Yeast extract, 2% Peptone, 2% Dextrose, and 0.004% adenine hemisulfate) at 30°C with constant shaking at 225 rpm to an OD A600  of 0.8-1.0.  30 x 10^7 cells were harvested by centrifugation and washed  with nuclease-free water.

extract_protocol:
Total RNA was isolated using RiboPure™-Yeast Kit (Life Technologies). To eliminate residual DNA, total RNA was treated with 2 μl DNase I (TURBO DNA-free™ Kit; Life Technologies) for 45 min at 37°C. RNA was digested once more using DNase I (RNase-Free DNase set; Qiagen) for 10 min at room temperature before purification using the RNeasy® Kit (Qiagen). Prior to library construction for RNA-seq, the quality of RNA was ascertained using Bioanalyzer (Agilent), and the RNA integrity number (RIN) for the samples ranged from 6.8−8.1.
Library construction is performed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero™ Magnetic Gold Kit (Yeast)  (cat# RS-122-2401, RS-122-2402) as described below.  Briefly, Ribosomal RNA is removed from total RNA samples (100 ng to 4 ug) using biotinylated Ribo-Zero oligos attached to magnetic beads that are complimentary to yeast cytoplasmic  and chloroplast rRNA. Following purification, the rRNA-depleted sample is fragmented with divalent cations under elevated temperatures and primed with random hexamers in preparation for cDNA synthesis.  First strand reverse transcription is accomplished using Superscript II Reverse Transcriptase (Invitrogen cat#18064-014).  Second strand cDNA synthesis is accomplished using DNA polymerase I and Rnase H under conditions in which dUTP is substituted for dTTP, yielding blunt-ended cDNA fragments in which the second strand with dUTP.  An A-base is added to the blunt ends as a means to prepare the cDNA fragments for adapter ligation and block concatamer formation during the ligation step.  Adapters containing a T-base overhang  are ligated to the A-tailed DNA fragments.  Ligated fragments are PCR-amplified (12-15 cycles) under conditions in which the pcr reaction enables amplification of the first strand cDNA product, whereas attempted amplifciation of the second strand product stalls at dUTP bases and therefore is not represented in the amplified library.  The PCR-amplified library is purified using Agencourt AMPure XP beads (Beckman Coulter Genomics cat#A63881).  Following amplification, the library is purified by bead based methodologies.  The concentration of the amplified library is measured with a NanoDrop spectrophotometer and an aliquot of the library is resolved on an Agilent 2200 Tape Station using a D1K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1K (cat# 5067-5363 and 5067-5364) assay to define the size distribution of the sequencing library.  Libraries are adjusted to a concentration of approximately 10 nM and quantitative PCR is performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to calculate the molarity of adapter ligated library molecules.  The concentration is further adjusted following qPCR to prepare the library for Illumina sequence analysis. The library was then subjected to 50bp single read sequencing in an Illumina Hiseq2000 sequencer.

value: set1_delta::KanMX Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta20 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Msi2 knockdown 3

city: La Jolla

first: Roman

last: Sasik

address: 9500 Gilman Dr.

department: Center for Computational Biology and Bioinformatics

institute: UCSD

description:
cells treated with shRNA for Msi2
MP2_Msi2_3

data_processing:
Basecalls performed using CASAVA version 1.4
Gene-level counts produced using kallisto version 0.42.1 (http://omictools.com/kallisto-s9409.html)
Genome_build: hg19
Supplementary_files_format_and_content: abundance measurement

source_name: pancreatic cancer cell line

growth_protocol: MIA PaCa2 cells were cultured in DMEM containing 10% FBS, 5% Horse serum, and 1% Pen/strep.

extract_protocol:
RNA was harvested from sorted cells using a Qiagen RNA micro kit. 200ng of total RNA was submitted for library generation and sequencing.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: MIA PaCa2 cells were treated with lentivruses containing shRNAs against scrambled, Musashi1 (viral GFP tag) or Musashi2 (viral Tomato Red tag). Cells were infected for 72 hours followed by sorting of GFP, Tomato Red, or double positive cells.

value: MIA PaCa-2


title: Set1Flag_3

description:
Set1_Flag_Plus.bw
Set1_Flag_Minus.bw
SET1 overexpression Repeat 3

value: ADH1p-FlagSET1::URA3 Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta20 ura3-52 HTA1-HTB1 (CEN HIS3)


title: jhd2_3

description:
Jhd2_Plus.bw
Jhd2_Minus.bw
jhd2∆  Repeat 3

value: jhd2_delta::KanMX Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta20 ura3-52 HTA1-HTB1 (CEN HIS3)


title: WT_3

description:
WT_Plus.bw
WT_Minus.bw
wild type Repeat 3

value: Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta20 ura3-52 HTA1-HTB1 (CEN HIS3)


title: wild type H3-2Flag ChIP repeat2

description: ChIP DNA

value: HHT1-V5-LoxP-HphMX-LoxP-2FLAG PTDH3-Cre-EBD78::URA3 (hht2-hhf2)delta::NatMX6 Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Set1G990E_2

description:
Set1_G990E_Plus.bw
Set1_G990E_Minus.bw
SET1-G990E overexpression Repeat 2

value: ADH1p-FlagSET1G990E::URA3 Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta20 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Set1Flag_2

description:
Set1_Flag_Plus.bw
Set1_Flag_Minus.bw
SET1 overexpression Repeat 2


title: Set1G990E_3

description:
Set1_G990E_Plus.bw
Set1_G990E_Minus.bw
SET1-G990E overexpression Repeat 3


title: Msi2 knockdown 1

description:
cells treated with shRNA for Msi2
MP2_Msi2_1


title: Msi1 knockdown 3

description:
cells treated with shRNA for Msi1
MP2_Msi1_3


title: 8V5-Set1 input

description: genomic DNA


title: jhd2D H3-V5 ChIP repeat2

description: genomic DNA


title: wild type H3-V5 ChIP repeat2

description: genomic DNA


title: Msi1 knockdown 1

description:
cells treated with shRNA for Msi1
MP2_Msi1_1


title: set1D H3-V5 ChIP repeat2

description: genomic DNA

value: set1delta::KanMX6; HHT1-V5-LoxP-HphMX-LoxP-2FLAG PTDH3-Cre-EBD78::URA3 (hht2-hhf2)delta::NatMX6 Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: jhd2D H3-2Flag ChIP repeat1

description: ChIP DNA


title: set1D H3-2Flag ChIP repeat2

description: ChIP DNA


title: Msi1 and Msi2 double knockdown 3

description:
cells treated with shRNAs for Msi1 and Msi2
MP2_M1M2_3


title: S_1058

value: Subcutaneous fat


title: jhd2_2

description:
Jhd2_Plus.bw
Jhd2_Minus.bw
jhd2∆  Repeat 2


title: set1_2

description:
Set1_Plus.bw
Set1_Minus.bw
set1∆  Repeat 2


title: Msi2 knockdown 2

description:
cells treated with shRNA for Msi2
MP2_Msi2_2


title: Set1G990E_1

description:
Set1_G990E_Plus.bw
Set1_G990E_Minus.bw
SET1-G990E overexpression Repeat 1


title: WT_2

description:
WT_Plus.bw
WT_Minus.bw
wild type Repeat 2


title: Msi1 and Msi2 double knockdown 1

description:
cells treated with shRNAs for Msi1 and Msi2
MP2_M1M2_1


title: wild type H3-2Flag ChIP repeat1

description: ChIP DNA


title: Set1Flag_1

description:
Set1_Flag_Plus.bw
Set1_Flag_Minus.bw
SET1 overexpression Repeat 1


title: Msi1 and Msi2 double knockdown 2

description:
cells treated with shRNAs for Msi1 and Msi2
MP2_M1M2_2


title: Msi1 knockdown 2

description:
cells treated with shRNA for Msi1
MP2_Msi1_2


title: jhd2_1

description:
Jhd2_Plus.bw
Jhd2_Minus.bw
jhd2∆  Repeat 1


title: control shRNA 2

description:
cells treated with control shRNA
MP2_Ctr2


title: control shRNA 3

description:
cells treated with control shRNA
MP2_Ctr3


title: set1D H3-V5 ChIP repeat1

description: genomic DNA


title: wild type H3-V5 ChIP repeat1

description: genomic DNA

value: HHT1-V5-LoxP-HphMX-LoxP-2FLAG PTDH3-Cre-EBD78::URA3 (hht2-hhf2)delta::NatMX6 Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: WT_1

description:
WT_Plus.bw
WT_Minus.bw
wild type Repeat 1


title: jhd2D H3-V5 ChIP repeat1

description: genomic DNA


title: control shRNA 1

description:
cells treated with control shRNA
MP2_Ctr1


title: set1D H3-2Flag ChIP repeat1

description: ChIP DNA


title: set1_1

description:
Set1_Plus.bw
Set1_Minus.bw
set1∆  Repeat 1


title: set1D nucleosome  rep3

data_processing: De-multiplexed Fastq files from the Illumina pipeline were aligned to the Saccharomyces cerevisiae genome release 64 (UCSC SacCer3) using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Alignments were converted to sorted, indexed Bam files using samtools. Separate sequence lanes from the same sample were merged using samtools.

source_name: MNase-digested mononucleosome

extract_protocol:
Cells were crosslinked for 20 minutes by addition of formaldehyde to 1% final concentration in the media and quenched by the addition of 1M glycine to 0.1M final concentration. 33 x 10^7 cells were harvested by centrifugation and washed twice with Phosphate-Buffered Saline and once with spheroplasting buffer (1M Sorbitol, 10 mM Tris.Cl pH 7.5, 15 mM 2-mercaptoethanol and protease inhibitors (PIs): 1 mM PMSF, 1μg/ml aprotinin, 1μg/ml leupeptin, 1μg/ml pepstatin A). Cross-linked cells were resuspended in 1ml fresh spheroplasting buffer before adding Lyticase (500 units; Sigma) and incubated in a 37oC water-bath shaker for 15-20 min with gentle agitation. Spheroplasts were washed twice with 25ml wash buffer (50mM Tris pH 7.5, 100mM KCl, 2.5mM MgCl2, 400mM Sorbitol, and PIs), resuspended in MNase digestion buffer (50mm Tris pH 7.5, 100mM NaCl, 5mM MgCl2, 1mM CaCl2 and 0.1% Triton X-100 and PIs) and digested with 10 units of MNase (Micrococcal Nuclease, Worthington Biomedical) at 37oC for 15 min. Digestion was stopped by adding 100μL 10X stop buffer (50mM Tris.Cl pH 7.5, 500mM NaCl, 100mM EDTA, 20mM EGTA, 10% Triton X-100 and 1% Sodium Deoxycholate). After centrifugation, soluble chromatin (500μL) was mixed with an equal amount of elution solution (1% SDS, 100 mM sodium bicarbonate) and NaCl (200mM final concentration), reverse cross-linked for 5h at 65oC and ethanol precipitated overnight at -20oC. After centrifugation, the DNA pellet was washed with cold 70% ethanol, air-dried, dissolved in nuclease-free water. RNase A (2μL of 10mg/ml stock, Qiagen) digestion was performed at 37oC for 30 min followed by Proteinase K digestion (2 μL, Roche) for 1h at 42oC in the presence 1X Proteinase K digestion buffer (10mM Tris.Cl pH 8.0, 5mM EDTA, 0.5% SDS). DNA was purified using Qiagen PCR purification kit and eluted in 50μL nuclease-free water. DNA concentration was measured using NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific).
Equal amount of purified DNA (10ng) was subjected to library construction using the NEBNext®  ChIP-Seq Library Prep Reagent Set for Illumina following the manufacturer's protocol. Adaptor-ligated and index primer-containing amplified DNA corresponding to mononucleosomes was resolved in an 1.5% agarose gel and purified using the Qiagen Gel Extraction Kit, except incubation to melt the gel was performed at 37oC for 30min. The library was then subjected to 50bp single read sequencing in an Illumina Hiseq2000 sequencer.

value: set1_delta::KanMX Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: jhd2D nucleosome  rep2

value: jhd2_delta::KanMX Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: set1D nucleosome  rep1


title: set1D nucleosome  rep2


title: wild type nucleosome  rep3

value: Mat a hta1-htb1D hta2-htb2D lys2-128deltahis3delta200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: wild type nucleosome  rep2

value: HTABWT


title: wild type H3K4me3 repeat2

description:
ChIP DNA
contains DNA from Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) used as calibration genome
calibrated processed data file: WT-K4ME3.calibrated.bw

data_processing:
De-multiplexed Fastq files from the Illumina pipeline were aligned using Novoalign version 2.8 (http://www.novocraft.com), allowing for one random repeat, base recalibration, and a minimum alignment quality score of 13. Sequences were aligned first to the C glabrata genome; unaligned read sequences were exported and re-aligned to the S cerevisiae genome. To identify orthologous alignments, aligned sequences to the C glabrata genome were exported as fastq and re-aligned to S cerevisae; sequences that aligned to both genomes were considered orthologous. Alignments were converted to sorted, indexed Bam files using samtools. Biological replicates, after comparison, had their alignments were merged using samtools.
Fragment coverage was generated using the BioToolBox bam2wig application (v1.35) by extending each alignment in the 3' direction by 200 bp and counting the coverage. Depth was normalized to reads per million (RPM). Coverage tracks were converted to bigWig files for visualization and analysis using UCSC utilities.
Replicates were compared by generating a Pearson correlation of the coverage files using the UCSC utility bigWigCorrelate with a max threshold of 250. Replicates were merged with samtools and coverage tracks re-generated.
The number of alignments to S cerevisiae and C glabrata were counted using samtools. The number of orthologous alignments were subtracted from C glabrata counts. A normalization factor was derived from the formula (Cg_Ref x Sc_ChIP) / (Sc_Ref x Cg_ChIP), where Cg_Ref is the number of Input alignments to C glabrata, Sc_ChIP is the number of ChIP alignments to S cerevisiae, Sc_Ref is the number of Input alignments to S cerevisiae, and Cg_ChIP is the number ChIP alignments to C glabrata. This normalization scale was then applied to every genomic position represented in a bedGraph file of the ChIP fragment coverage to S cerevisiae using the script manipulate_wig (https://github.com/tjparnell/HCI-Scripts).
Genome_build: SacCer3
Genome_build: Candida glabrata, GCA_000002545.2, Ensembl Fungi release 30
Supplementary_files_format_and_content: BigWig files representing uncalibrated fragment (uniformly extended to 200 bp) coverage depth normalized to reads per million mapped. Filenames have uncalibrated in their name.
Supplementary_files_format_and_content: BigWig files representing the the merged replicate, calibrated fragment coverage depth. Filenames have calibrated in there name. These files are available in a tar archive on the series record.

organism:
Saccharomyces cerevisiae
Nakaseomyces glabratus

source_name: sonicated soluble chromatin

extract_protocol:
Yeast strains were grown to mid-log phase and cross-linked with 1% formaldehyde for 20 min at room temperature. Crosslinking was stopped using 0.125 mM glycine and cells were washed with 1X PBS. Multiple aliquots of 40 × 107 cross-linked cells each were prepared. Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) was grown at 25°C and subjected to formaldehyde crosslinking as described for budding yeast, and individual aliquots containing 20 × 107 cells each were harvested. Cross-linked S. cerevisiae (40 × 107) and C. glabrata (20 × 107) cells were mixed together in FA140+SDS buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl, 0.1% SDS) and lysed by bead beating. Lysates from two sets of S. cerevisiae and C. glabrata admixture (i.e., 120 × 107 cells in total) were pooled and sonicated in a Diagenode Bioruptor™ for 30 min (6 cycles of 5 min each, 30 sec ON and 30 sec OFF at high setting). Soluble chromatin was obtained after two sequential high-speed centrifugations of the sonicated lysate (16,100 X g for 5 min and 15 min). SDS was removed by dialysis as follows. Soluble chromatin obtained from four batches of control or mutant budding yeast and candida admixture were pooled, transferred to a 5 ml Float-A-Lyzer G2 dialysis device (3.5 - 5 kD MWCO) (Spectra/Por) and dialyzed initially for 2 h in 1 L FA140 buffer followed by an overnight dialysis in fresh 4 L FA140 buffer. Dialysate was centrifuged at 16,100 X g for 15 min at 4°C. A 100 μl aliquot was set aside for isolating input DNA. The remaining dialysate was split equally into three tubes and pre-cleared using 50 μl blocked Dynabeads® Protein A for 1h at 4°C with end-over-end rotation. Dynabeads® Protein A were blocked with BSA and fish skin gelatin as described above except bead blocking was done for 4 h. Pre-cleared lysate was combined with either α-H3K4me1 (20 μl, Active Motif) or α-H3K4me2 (10 μl, Millipore) or α-H3K4me3 (10 μl, Active Motif) antibody and 100 μl blocked Dynabeads® Protein A, and incubated overnight at 4°C with end-over-end rotation. Beads were initially washed with three times with FA140 buffer by inversion followed by sequential washes (5 min each with end-over-end rotation): FA140 buffer (2 times), FA1000 buffer (2 times), FA500 (2 times), LiCl/NP40 buffer (1 time) and TE (1 time). ChIP DNA was eluted in 400 μl elution solution (1% SDS, 100 mM sodium bicarbonate) and incubated at room temperature with end-over-end mixing for 40 min. The eluate is transferred to a fresh 0.65 ml tube, 18 μl 5 M NaCl is added and mixed well. At this stage, 400 μl elution solution and 18 μl 5 M NaCl is also added to the 50 μl soluble chromatin set aside to isolate ‘‘input’’ DNA. Reverse crosslinking is done by incubation at 65 °C for 5 h in a thermal cycler. The solution was transferred to a fresh 1.5 ml tube, 1 ml ethanol was added, mixed well and precipitation was allowed to occur overnight at -20 °C. The samples were centrifuged at maximum speed in a table-top centrifuge at 4°C. The pellet was washed with 70% ethanol followed by centrifugation at high speed. The pellet was dissolved in 90 μl water, treated with 2 μl 10 mg/ml RNase A and followed by an incubation at 37 °C for 30 min. Proteins were removed by adding 10 μl 10X PNK buffer (100 mM Tris.Cl, pH 8.0, 50 mM EDTA, 5% SDS), 1 μl Proteinase K (Sigma) and incubating at 42°C for 1 h. DNA was purified using Qiaquick PCR Purification kit after addition of 20 μl 3 M Sodium acetate to adjust the pH. DNA was eluted in 50 μl water and stored at -20 °C. DNA concentration was measured using Qubit Fluorometer. Equal amount ChIP or input DNA was used in library construction using NEBNext® Multiplex Oligos (Index Primers Set 1 and Index Primer Set 2) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina.
Equal amount of purified DNA (10ng) was subjected to library construction using the NEBNext®  ChIP-Seq Library Prep Reagent Set for Illumina following the manufacturer's protocol. Adaptor-ligated and index primer-containing amplified DNA were resolved in an 2.0% agarose gel and 300-500 bp sized fragments were purified using the Qiagen Minelute Gel Extraction Kit, except incubation to melt the gel was performed at 37oC for 30min.

value: Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta_200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: wild type input repeat2

description:
genomic DNA
contains DNA from Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) used as calibration genome
calibrated processed data file:


title: jhd2Δ H3K4me2 repeat2

description:
ChIP DNA
contains DNA from Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) used as calibration genome
calibrated processed data file: Jhd2-K4ME2.calibrated.bw

value: jhdelta2_delta::KanMX Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta_200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Set1G990E input repeat2

value: AdeltaH1p-FlagSET1G990E::URA3 Mat a hta1-htb1delta hta2-htb2delta lys2-128deltahis3delta_200 ura3-52 HTA1-HTB1 (CEN HIS3)


title: Set1G990E H3K4me2 repeat2

description:
ChIP DNA
contains DNA from Candida glabrata (Anderson) Meyer et Yarrow Strain Designation: NCYC 388 (ATCC® 36909) used as calibration genome
calibrated processed data file: G990E-K4ME2.calibrated.bw


title: jhd2Δ input repeat2


title: jhd2D nucleosome  rep3