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Sample GSM3756688

Query DataSets for GSM3756688

Status

Public on May 10, 2019

Title

TBP-ChEC Nrd1-AA_drpd3_+Rap_rep2

Sample type

SRA

Source name

TBP-ChEC Nrd1-AA_drpd3_+Rap

Organism

Saccharomyces cerevisiae

Characteristics

strain background: HHY168
genotype/variation: Nrd1-AA drpd3
treatment: Plus Rapamycin 1h (1μg/ml)
sample group: Nrd1-AA drpd3 TBP-ChEC

Treatment protocol

With or without 1μg/ml of rapamycin for 1h

Growth protocol

All strains were derived from the Anchor-Away genetic backgrounds (Haruki et al. 2008). Cells were grown in YEPD medium (1% yeast extract, 1% peptone) supplemented with 2% glucose as carbon source. All strains were grown at 30°C and were not affected in growth by the different tags or deletions. Anchor-away of Nrd1-AA was induced by adding 1μg/ml of rapamycin to the medium.

Extracted molecule

genomic DNA

Extraction protocol

The experiment was performed as described in (Zentner et al., 2015) after some modifications. The yeast strains were cultured the day before in YEPD medium. They were diluted to OD600 of 0.2 in YEPD medium in the morning. The 60 mins rapamycin treatment was performed at OD600 of 0.4. For each condition, 50 mL cell culture was harvested at room temperature. The cells were washed twice in 1 mL of Buffer A (15 mM Tris-HCl pH 7.5, 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine and protease inhibitor tablet (Sigma-Aldrich). Cells were resuspended in 594 uL of buffer A and 6 uL of 10% digitonin (0.1% final concentration) were added to permeabilize the cells during 5min at 30 °C. Then CaCl2 was added (final concentration 5 mM) to activate the MNase cleavage. 200 uL were collected at 30s for SPT15-MNase or 20s for STH1-MNase and were immediately mixed with 200 uL of 2X Stop solution (400 mM NaCl, 20 mM EDTA, 4 mM EGTA, 1% SDS). The cells were treated with Proteinase K (0.4 ug/uL final concentration) and incubated at 55 °C for 30 minutes. The DNA was extracted using phenol:chloroform:isoamyl extraction, followed by precipitation by adding 30 ug glycogen, 500 uL of 100% ethanol and incubated at -20 °C for one hour. RNAse treatment was performed by adding 34.5 uL of Tris, pH 8.0 and 10 ug of RNAse per reaction. For size selection of DNA fragments, 75 uL of solid phase reversible immobilization beads (SPRI, AmpureXP Beckman Coulter) were added in 25 uL of RNAse treated DNA and the reaction was mixed by pipetting up and down 10 times. The beads were incubated at room temperature for 5 minutes. The tubes were placed in the magnetic rack and supernatant was transferred to the new tube containing 10 mM Tris and 0.2 M NaCl for each reaction. The DNA was extracted with phenol:chloroform:isoamyl solution and precipitated with 100% ethanol. The pellets were washed with 70% ethanol and resuspended in 29 uL of Tris, pH 8.0. DNA concentration was measured using Qubit dsDNA HS assay kit (Invitrogen) using 4 uL sample. The rest 25 uL was used to prepare sequencing libraries using NEBnext Ultra DNA library prep kit for Illumina (NEB). The samples were sequenced in the paired-end mode at the iGE3 genomics sequencing platform in Geneva.
NEBnext Ultra DNA library prep kit for Illumina (NEB)

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

NOTE: processed data was generated from both rep1 & rep2, and is linked to the corresponding rep1 sample records.

Data processing

library strategy: Chromatin endogenous cleavage (ChEC)-seq
Paired-end reads were aligned to sacCer3 genome assembly using Bowtie2 (Langmead and Salzberg, 2012; Langmead et al., 2009) with default options from the Galaxy server (Afgan et al., 2018). PCR duplicates were removed from the analysis. DeepTools 2.0 (Ramirez et al., 2016) was then used through the bamCoverage function with size selection of fragments (0-120bp, 0-120bp and 120-200bp for TBP-ChEC, ATAC-seq and Sth1-ChEC respectively) and counting of only the 3bp at the center of fragments.
Genome_build: saccer3
Supplementary_files_format_and_content: bigWig files corresponding to the mean of rep1 and rep2. cpm normalization.

Submission date

May 09, 2019

Last update date

May 10, 2019

Contact name

Julien Soudet

E-mail(s)

julien.soudet@unige.ch

Organization name

Université de Genève

Department

BICEL