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Sample GSM4696191 Query DataSets for GSM4696191
Status Public on Jul 26, 2021
Title ChIPexo_Rpb3_WT_R1
Sample type SRA
 
Source name Yeast cell extract
Organism Saccharomyces cerevisiae
Characteristics strain name: yFR116
genotype: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3
chip antibody: anti-Rpb3 (BioLegend 665004, 1.5 μL)
Treatment protocol Cultures were crosslinked with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 30 min. Crosslinking was quenched with 125 mM glycine.
Growth protocol Cells were grown at 30°C and 200 rpm in YPD (yeast extract-peptone-2% glucose, supplemented with 44 µM adenine) medium as follow. Strains were streaked from glycerol stocks onto 2% agar YPD plates and grown at 30°C for 2-3 days. An isolated colony was then grown overnight in 10 mL of YPD. This preculture was used to inoculate 50 mL of YPD at an OD600 of 0.1 which was grown to an OD600 of 0.7-0.9.
Extracted molecule genomic DNA
Extraction protocol ChIP-exo experiments were performed essentially as described previously (Rossi et al., 2018) with some modifications. After following our standard ChIP protocol using a crosslinked 50 mL cell culture at an OD600~0.8 per IP and the appropriate antibody-coupled Dynabeads (see above), beads were washed twice with Lysis buffer, twice with Lysis buffer 500, twice with Wash buffer and once with 10mM Tris-HCl pH8.0. Beads were then briefly centrifuged to remove all liquid before being used in the ChIP-exo library as follows. For A-tailing of the immunoprecipitated DNA, beads were resuspended in 50 μL of 1X NEBuffer 2 (NEB, B7002S) containing 0.1 mM dATP and 15 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M) and incubated at 37˚C for 30 min. After washing the beads with 10 mM Tris-HCl pH 8.0, dA-tailed DNA was ligated to the First adapter by resuspending the beads in 45 μL of 1X Rapid ligation buffer (Enzymatics, B1010) containing 375 nM of First ligation adapter (synthesized by IDT; see Table S2 for a list of adapters with their bar codes), 10 units of T4 polynucleotide kinase (NEB, M0201S) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L) and incubating at room temperature for 60 min. After washing the beads with 10 mM Tris-HCl pH 8.0, the ligated DNA was subjected to a Fill-in reaction by resuspending the beads in 40 μL of 1X phi29 buffer (NEB) containing 0.2 mg/mL BSA (NEB), 180 μM dNTPs and 10 units of phi29 DNA polymerase (NEB, M0269S) and incubating at 30˚C for 20 min. After washing the beads with 10 mM Tris-HCl pH 8.0, DNA was subjected to λ exonuclease digestion by incubating the beads with 50 μL of λ exonuclease reaction buffer (NEB) containing 0.1% Triton X-100, 5% DMSO and 10 units of λ exonuclease (NEB, M0262S) at 37˚C for 30 min. To elute and reverse-crosslink DNA, beads were resuspended in 40 uL of reverse crosslink mix containing 25 mM Tris-HCl pH7.5, 200 mM NaCl, 2 mM EDTA, 0.5% SDS and 30 μg of Proteinase K (Thermo Scientific, 25530049) and incubated at 65˚C for 16 hr. Eluted DNA was then subjected to a 1.8X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions before proceeding with Second adapter ligation as follows. Beads were resuspended in 40 μL of 1X T4 DNA ligase reaction buffer (NEB, B0202S) containing 375 nM of Second ligation adapter (synthesized by IDT; see Table S2) and 1,200 units of Rapid T4 DNA ligase (Enzymatics, L6030-HC-L), and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1.8X cleanup with KAPA Pure Beads and eluted in 15 μL ddH2O. Libraries were PCR-amplified in 40 μL reactions with 15-18 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and 500 nM of each primer P1.3 and P2.1 (synthesized by IDT; see Table S2). At this step, 10 μL from each sample was further amplified for six more cycles to visualize the quality of the library by electrophoresis on a 2% TAE-agarose gel stained with ethidium bromide. Once the library validated, DNA was subjected to a 1X cleanup with KAPA Pure Beads, followed by a double size selection (0.6X-1X) leading to DNA fragments in the 150-450 bp range. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library were pooled (14-16 libraries per pool) and subjected to sequencing on an Illumina HiSeq 4000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID #1: 1st ligation adapter (D702)
Barcode ID #2: 2nd ligation adapter (D501)
ChIP-exo
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
 
Description Biological replicate 1 of 2. RNAPII (Rpb3) occupancy by ChIP-exo in WT cells. Rpb3 ChIP-exo was performed using a rabbit polyclonal antibody against Rpb3.
processed data file (combined biological replicates): ChIPexo_Rpb3_yFR116WT_CJ2-CJ6_20190412.bw
Data processing Paired-end reads from ChIP-exo data were aligned to the yeast S. cerevisiae genome (sacCer3 from UCSC Genome Browser) using the BWA version 0.7.17 (Li, 2013). Unmapped reads, reads not mapped properly in pairs, and duplicates were removed from the analysis using SAMtools version 1.8 (Li et al., 2009). For each paired-end read, only the first read was kept for further analysis. The aligned files were converted to BED using BEDTools version 2.28.0 (Quinlan and Hall, 2010). BED files containing reads from replicates were further analyzed individually in addition to being merged. The reads were moved 6 bp towards the 3’ end to correct for the offset of exonuclease stops and the site of crosslinking using a custom java script called bed-tools-j, version 2.1 – https://github.com/francoisrobertlab/bed-tools-j/releases/tag/2.1. Three genome coverage files were computed using the 5’ position of the moved reads using BEDTools. Coverage files were generated for reads mapping the positive strand, the negative strand, and the merged strands. The genome coverage files were converted to the bigWig format using the utilities from UCSC Genome Browser (Casper et al., 2018). The analysis was partially automated using a custom script called seqtools available at https://github.com/francoisrobertlab/seqtools.
For each biological replicate, three genome coverage files (bigWig) for reads mapping the positive strand (ending with "pos.bw"), the negative strand (ending with "neg.bw"), and the merged strands (ending with ".bw"). In addition, a coverage file (merge of both strands) is provided for the combined biological replicates (beginning with "ChIPexo").
Genome_build: S. cerevisiae (UCSC sacCer3)
Supplementary_files_format_and_content: bigWig
 
Submission date Jul 27, 2020
Last update date Jul 26, 2021
Contact name Francois Robert
E-mail(s) francois.robert@ircm.qc.ca
Organization name IRCM
Lab Chromatin and Genomic Expression
Street address 110 av des Pins Ouest
City Montreal
State/province QC
ZIP/Postal code H2W 1R7
Country Canada
 
Platform ID GPL21656
Series (2)
GSE155138 FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [ChIP-exo]
GSE155144 FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner
Relations
BioSample SAMN15649455
SRA SRX8829409

Supplementary file Size Download File type/resource
GSM4696191_CJ2_ChIPexo_Rpb3_yFR116WT_R1_20190412.bw 24.6 Mb (ftp)(http) BW
GSM4696191_CJ2_ChIPexo_Rpb3_yFR116WT_R1_20190412_neg.bw 18.1 Mb (ftp)(http) BW
GSM4696191_CJ2_ChIPexo_Rpb3_yFR116WT_R1_20190412_pos.bw 18.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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