|
| Status |
Public on Dec 17, 2021 |
| Title |
25min-H3K4me3-ChIPNChAP-Mcm23A-2readsFigS7BGEO |
| Sample type |
SRA |
| |
|
| Source name |
Mcm23A mutant yeast derived from W303 with the h-ENT1 nucleotide transporter and HPV-Thymidine kinase and HA tagged Rpb3 (subunit of RNAPol2) and mcm2-3A::HYG that replaced the endogenous mcm40
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: Mcm23A mutant
cell cycle: mid early S-phase (34% of the genome is unreplicated)
edu incubation: from beginning of release from G1 arrest
antibody: H3K4me3
|
| Treatment protocol |
cell aliquots were fixed with 1% (w/v) formaldehyde for 15 min at 30°C followed by 5 min of quenching in 125 mM Glycine. Cell pellets were then washed with cold PBS and flash frozen in liquid nitrogen and kept at -80°C until further processing.
|
| Growth protocol |
Cells were grown overnight at 30°C in SCD-URA. The culture was diluted to OD600 ~0.3 the next morning and grown to OD600 ~0.65 and re-diluted to OD600 ~0.3 in fresh media. The culture was synchronized with the addition of 0.15 μg/ml α factor for 3h30min at 30°C. Cells were released from arrest in preheated (SCD-URA) + 10 μM EdU and grown at 30°C for indicated times before fixation with formaldehyde.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked frozen cell pellets were re-suspended in 1.5 ml NP lysis buffer (100 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 10% NP-40, 50 mM EDTA, 0.1 % SDS (optional), 1 mM PMSF and 1xEDTA-free protease inhibitor cocktail (Roche)). The suspension was then split into aliquots each containing ~billion cells. Zirconium Sillicate beads (400 μl, 0.5 mm) were then added to each aliquot and cells were mechanically disrupted using a bullet blender (Next Advance) for 4 times x 3 min (intensity 8). Spheroplasts were treated with micrococcal nuclease or sonicated. Sonicated or digested chromatin was subject to ChIP with the indicated antibody. An aliquot was set aside for the input DNA fraction prior to ChIP. EdU incorporated into ChIPed and Input DNA fragments was biotinylated using Click chemistry and nascent DNA was purified with streptavidin coated beads (ChIP-NChAP and NChAP fractions, respectively). The supernatant after binding to streptavidin beads was kept for the unreplicated ChIP fraction and an input DNA and a bulk ChIP DNA aliquot were set aside prior to biotinylation for the Input and ChIP fractions.
DNA samples from all fractions were treated with calf intestinal phosphatase (NEB). NGS libraries were constructed either with costum enzymes( blunt-ended, A-tailed, and ligated (Epicentre) to Illumina genomic PE adaptors with custom 5bp inline barcodes (first 5bp in raw data read)) or using the TrueSeq V2 LT Sample prep kit (Illumina) with the Illumina unique multiplexing indexes Set A and B. The custom library samples were amplified with the Phusion enzyme (NEB) for 18 PCR cycles with Illumina PE1 and PE2 primers. The primer mix from the kit was used in the PCR amplification step (also with Phusion) for the samples prepared with TrueSeq V2 LT (15 cycles). For nascent (replicated NChAP and ChIPNChAP fractions) libraries, the nascent 'EdU containing) strand was separated from its complement with primer extension in the presence of dUTP and the dUTP strand was then digested with the USER enzyme cocktail prior to PCR amplification. All the steps for nascent libraries were done on streptavidine coated magnetic beads.
Strand specific MNase-CHIP-seq or strand specific sonicated-ChIP-seq of replicated DNA fragments. The method which we call ChIP-NChAP (Nascent Chromtin Avidin Pulldown) was developed in the Radman-Livaja group.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
H3K4me3 ChIP followed by nascent DNA purification by EdU-biotin pulldown from S-phase 25min after G1 arrest with alpha factor
all_readsFandR_norm_50bp_Figs_6_S7BGEO.txt
Sonicated chromatin,25min after release into S phase, H3K4me3 on replicated DNA.paired end reads, replicate 2
|
| Data processing |
Illumina OLB-1.8.0/CASAVA_v1.7.0 were used for base calling
time points and time point fractions were separated either by in line barcodes and the 5bp barcode were removed from the read sequence and saved into separate fastq files from a unique fastq file obtained from the sequencing service using a custom perl script, or with Illumina unique indexes (Set A and B) and demultiplexed after sequencing into separate fastq files by the sequencing facility
reads were mapped to the yeast S288C genome using BLAT
Paired end read count distribution in 50bp windows was determined separately for Watson (marked as F in the processed data files) and Crick (marked as R in the processed data files) strands
the genome average read count value was calculated and the data were normalized to one
Genome_build: S288C (S. cerevisiae)
Supplementary_files_format_and_content: tab-delimited text files of indicated feature enrichment (columns) normalized to the average genome read count and distributed in 50bp bins. 50bp bins marked with R indicate repetitive sequences.
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| |
|
| Submission date |
Oct 27, 2020 |
| Last update date |
Dec 17, 2021 |
| Contact name |
Marta Radman-Livaja |
| E-mail(s) |
mrl5374@gmail.com
|
| Phone |
+33434359667
|
| Organization name |
CNRS
|
| Department |
IGMM
|
| Street address |
1919 route de Mende
|
| City |
Montpellier |
| ZIP/Postal code |
34293 |
| Country |
France |
| |
|
| Platform ID |
GPL26302 |
| Series (2) |
| GSE160262 |
The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [ChIP-seq] |
| GSE160509 |
The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand |
|
| Relations |
| BioSample |
SAMN16575765 |
| SRA |
SRX9381308 |
