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Sample GSM7529862

Query DataSets for GSM7529862

Status

Public on Feb 03, 2024

Title

Input_rpb1_OFF

Sample type

SRA

Source name

Schizosaccharomyces pombe

Organism

Schizosaccharomyces pombe

Characteristics

cell type: Schizosaccharomyces pombe
strain: Sp972
genotype: LY7386
treatment: 20uM Thiamine for 3hrs at 32degreeC (from t=0 to t=3). At t=2,5, added 100nM 5aIAA in NaOH.

Treatment protocol

Degron-tagged strains were treated with 100nM 5-adamantyl-IAA for 1hr before the end of the arrest (Dhp1-3xsAID) or 30mn before the end of the arrest (Rpb1-3xsAID).

Growth protocol

Cells were grown in Pombe Minimal Glutamate media at 32°C and arrested in mitosis by a thiamine repressible system leading to APC inhibition (nmt41-slp1) at 32°C for a total of 3hours with 20uM thiamine.

Extracted molecule

genomic DNA

Extraction protocol

Cells were fixed with 1% (ChIP) or 3% (Hi-C) formaldehyde 5mn at 32°C, 20mn at 19°C and quenched 5mn with 125mM glycine before being put on ice, washed with 1X PBS and frozen in liquid nitrogen. For ChIP, fixed cerevisae cells with Psm3-GFP tagged cohesin were added and cells were lysed with precellys, sonicated with S220 covaris, clarified twice by centrifugation and subjected to overnight immunoprecipitation before washes. DNA was eluted and purified with Qiagen Quiaquick columns. For Hi-C, cells were lysed with precellys, digested with DpnII overnight, filled in with dTTP, dGTP, dCTP and biotin-14-dATP for 45mn at 37°C, ligated for 4hr at 25°C and crosslinks were reversed overnight, and DNA was recovered with Phenol/Chloroform/IAA extraction and Amicon 30K column washes.
For ChIPseq, NEBNext UltraII DNA Library Prep Kit E7645 and adapters E6609 were used. For Hi-C, libraries were performed with separate reagents, removing biotin from unligated ends with Klenow DNA Polymerase I, sonicating with Covaris, repairing extremities, A-tailing libraries and ligated NextFlex Adapters (CAT NOVA 514102)
Libraries were sequenced with pairedend sequencing 150bp on a Novaseq6000

Library strategy

Hi-C

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

For calChIPseq, an nf-core modified pipeline was used (https://www.biorxiv.org/content/10.1101/2022.03.18.484892v2) to generate normalized bigwig files
For Hi-C, raw reads were merged with the cat command and hicstuff version 3.1.2 was used to generate the 2D maps and the distance law plots by iterative mapping using bowtie2 with enzyme set as DpnII. Insulation scores were determined with cooltools.
Assembly: For Hi-C, the pombeASM294v2 genome was used but the mitochondrial chromosome was removed. For calChIPseq, chromosome II right arm was extended to include a partial telomeric sequence as described previously (https://www.biorxiv.org/content/10.1101/2022.03.18.484892v2)
Supplementary files format and content: Hi-C Matrices were converted to cooler format (.cool) with hicstuff for use in downstream analysis.
Supplementary files format and content: Bigwig files (.bigwig) for the Ips were bp normalized and corrected for calibration as described previously (https://www.biorxiv.org/content/10.1101/2022.03.18.484892v2) for downstream analysis.

Submission date

Jul 03, 2023

Last update date

Feb 03, 2024

Contact name

Léonard Colin

E-mail(s)

[email protected]

Organization name

ENS de Lyon

Department

LBMC