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Sample GSM4159627

Query DataSets for GSM4159627

Status

Public on Nov 13, 2019

Title

ATAC_T42_SD_T42S2

Sample type

SRA

Source name

Cerebral cortex tissue

Organism

Mus musculus

Characteristics

ztime: 18
time: 42
condition: SD
batch: 1
7 days after sd: NA
strain: C57BL/6J
gender: male

Treatment protocol

Mice for tissue collection were divided into two experimental cohorts, sleep deprived (SD) and non-sleep deprived (controls, Ctr). After a one-week habituation to the experimental setting, at the age of 11-12 weeks, the SD mice were sleep-deprived by gentle handling for 6 hours starting at light onset (zeitgeber time ZT0-ZT6), and allowed to recover according to the tissue collection schedule.

Growth protocol

Mice were housed under standard conditions according to the Swiss Animal Protection Act, under a 12h:12h light/dark cycle, with food accessible ad libitum at all times.

Extracted molecule

genomic DNA

Extraction protocol

Mice were anesthetized with isoflurane prior to decapitation. Cortex was rapidly dissected and flash frozen in liquid nitrogen. Frozen cortex of each individual was ground in liquid nitrogen and stored at -80°C until further use. Tissue from each mouse was distributed to the two protocols (RNAseq and ATAC-seq), such that both datasets originate from the exact same set of individuals.
Total RNA was extracted using the miRNeasy kit (Qiagen; Hilden, Germany) following the manufacturer's instructions. RNA-seq libraries were prepared using 1000 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina; San Diego, CA, USA) on a Sciclone liquid handling robot (PerkinElmer; Waltham, MA, USA) using a PerkinElmerdeveloped automated script. Libraries were sequenced on the Illumina HiSeq 2500 sequencer, producing >36 million (median 55 million) mappable single-end 100 bp reads. ATAC-seq was performed as followed, 100'000 nuclei were treated with 2.5 μl Tagment DNA enzyme (Nextera DNA Sample Preparation Kit, Illumina) in transposition buffer (10mM Tris Base, 5mM MgCl2, 10% DMSO, pH 7.6) at 37°C for 30 minutes, followed by cleanup on a Qiagen Minelute column. Fragments >1kb in size were removed using 0.6X, then 1X, volumes of AmpureXP beads (Beckman Coulter Life Sciences; Indianapolis, IN, USA). DNA fragments were subjected to 11 cycles of PCR amplification with Nextera dual index primers (Illumina) and the NEBNext High Fidelity 2X PCR Master Mix (New England Biolabs; Ipswich, MA, USA). PCR reactions were cleaned up with one volume AmpureXP beads, quantified by Qubit (ThermoFisher Scientific; Waltham, MA, USA) and quality controlled by Fragment Analyzer (Advanced Analytical Technologies; Ankeny, IA, USA). Libraries were sequenced on the Illumina HiSeq 2500 sequencer, producing >25 million (median 41 million) mappable 50 bp paired-end reads per sample after removal of duplicate and mitochondrial sequences.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Quantification of chromatin acessibility in mouse cortex (12:12 Light-Dark). Sample taken after sleep deprivation. (Time: 42 CTime: 18 )

Data processing

RNA-seq data were processed using the Illumina Pipeline Software version 1.82. All RNA-seq samples passed FastQC quality thresholds (version 0.10.1) and could thus be used in subsequent analysis.
Transcript abundance was quantified by kallisto version 0.43.0 (56) using the GRCm38 reference transcriptome (mm10) and the parameters --single -l 100 -s 20 -b 100. The abundances were processed as follows using sleuth version 0.29.0: transcript abundances were merged into gene counts in transcripts per million (TPM), after which we applied a detection cutoff of 5.5 on the mean gene counts across samples in the time series. Batch effects were corrected by ComBat.Transcript abundance was quantified by kallisto version 0.43.0 (56) using the GRCm38 reference transcriptome (mm10) and the parameters --single -l 100 -s 20 -b 100. The abundances were processed as follows using sleuth version 0.29.0: transcript abundances were merged into gene counts in transcripts per million (TPM), after which we applied a detection cutoff of 5.5 on the mean gene counts across samples in the time series. Batch effects were corrected by ComBat.
ATAC-seq reads were aligned to the mouse genome (mm10) using bowtie2 in paired-end mode, with the parameters recommended for open chromatin (--very-sensitive --maxins 2000 --no-mixed --no-discordant). Duplicatesequences were removed using samtools rmdup.
ATAC-seq data files were processed before peak calling as follows. Alignment files were converted into bed files and tags were extracted using bedtools version 2.26.0. Each tag position was shifted +4 base pairs on the positive strand and - 5 base pairs on the negative strand to center tags on transposase binding events.
The peak calling was performed on pooled tags for replicates using Macs2 version 2.1.1 [--nomodel --shift -75 --extsize 150], and peaks were filtered using a 0.05 FDR cutoff for a local random Poisson distributed background noise, captured by Macs2.
Peak boundaries were merged between time points and conditions in order to build a common peak mapping reference covering all samples. Finally, peak coverage was quantified using HTSeq version 0.6.1 for each sample, using the common mapping reference. We filtered low coverage peaks using a minimum mean threshold of 10 reads per peak.
Genome_build: mm10
Supplementary_files_format_and_content: Chromatin accessibility (peak coverage) [CPM], Coordinate of ATAC peaks, Gene expression [log2 TPM]

Submission date

Nov 13, 2019

Last update date

Nov 20, 2019

Contact name

Maxime Jan

E-mail(s)

maxime.jan@unil.ch

Organization name

University of Lausanne

Department

CIG