 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 26, 2021 |
| Title |
MNase-ChIP_Pob3_yFR930chd1D_R1 |
| Sample type |
SRA |
| |
|
| Source name |
Yeast cell extract
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain name: yFR930
genotype: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3, chd1{delta}::URA3
chip antibody: anti-Pob3 (a gift from T. Formosa, 1 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 20 mL of YPD. This preculture was used to inoculate 500 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 |
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries. Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers’ instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit. 1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads.
Barcode ID:Adapter 15 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001)
MNase-ChIP-Seq
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Biological replicate 1 of 2. Pob3 occupancy by MNase-ChIP-seq in chd1D cells. Pob3 MNase-ChIP-seq was performed using a rabbit polyclonal antibody against Pob3.
processed data file (two biological replicates combined): MNaseChIP_Pob3_yFR930chd1D_CJ3-CJ4comb_20200319.bw
|
| Data processing |
Paired-end reads from MNase-ChIP-seq data where aligned on the S. cerevisiae genome version sacCer3 using Bowtie2 version 2.3.4.3 (Langmead and Salzberg, 2012) with the parameter “-X 1000” to include longer fragments. Unmapped and low-quality fragments were removed using SAMtools version 1.9 using the following parameters “view -f 2 -F 2048”. Duplicates were removed using SAMtools with the following parameter “fixmate -m” and then “markdup -r”. The aligned files were converted to BED using BEDTools version 2.27.1 using a custom software called seqtools version 1.0 available at https://github.com/francoisrobertlab/seqtools with the command “seqtools bam2bed” resulting in BED files containing the DNA fragments. The BED files of replicates were merged using seqtools with command “seqtools merge”. Genome coverage files were generated using customs scripts “seqtools” with commands “mnasetools prepgenomecov” and “seqtools genomecov” that require BEDTools version 2.27.1 and KentUtils (downloaded on July 16th 2018). The genome coverage files were converted to the bigWig format using the utilities from UCSC Genome Browser (Casper et al., 2018).
Genome_build: S. cerevisiae (UCSC sacCer3)
Supplementary_files_format_and_content: bigWig files for both individual replicates (beginning with "CJ") and the combined biological replicates (beginning with "MNase").
|
| |
|
| Submission date |
Jul 27, 2020 |
| Last update date |
Jul 26, 2021 |
| Contact name |
Francois Robert |
| E-mail(s) |
[email protected]
|
| 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 |
GPL27812 |
| Series (2) |
| GSE155143 |
FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner [MNase-ChIP-Seq] |
| GSE155144 |
FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner |
|
| Relations |
| BioSample |
SAMN15649492 |
| SRA |
SRX8829459 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4696255_CJ3_MNase-ChIP_Pob3_yFR930chd1D_R1_20200319.bw |
30.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
