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Sample GSM3193421

Query DataSets for GSM3193421

Status

Public on Feb 26, 2019

Title

chipseq_FF_rep3_input

Sample type

SRA

Source name

postnatal testis

Organism

Mus musculus

Characteristics

tissue: postnatal testis
developmental stage: developmental synchronization to enrich for preleptotenes
strain: C57BL/6 (<4% 129)
sorting: no sorting
genotype: Stra8 FLAG/FLAG
Sex: male
sample type: Input chromatin control for ChIP-seq sample
library prep kit: Accel-NGS 2S Plus DNA Library Kit (Swift Biosciences)
extract protocol: ChIP protocol: Samples were fixed and sonicated, and the chromatin was immunoprecipitated with the FLAG antibody. For each ChIP sample, a small fraction of the sonicated chromatin was saved as input chromatin control. Crosslinking was reversed, samples were treated with RNaseA and proteinase K, and the DNA was purified.

Treatment protocol

Mouse testes enriched for meiosis-initiating germ cells were obtained by synchronizing spermatogenesis in vivo by the WIN18,446/RA protocol. Male mice were treated with WIN18,446 daily from postnatal day (P) 2 to P8, and they were treated with retinoic acid on P9. Testes were collected ~6.75 days after the RA injection. A small portion of the testis was reserved for histology to confirm successful synchronization and the enrichment of preleptotene cells (germ cells initiating meiosis).

Extracted molecule

genomic DNA

Extraction protocol

Sequencing libraries were prepared using commercial kits.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

synchronizedtestis_Stra8-FLAGFLAG_FLAGchipseq_3_input

Data processing

For ChIP-seq: FASTX Toolkit v0.0.14 was used to filter out low-quality sequencing reads and to trim reads to 40 bp.
For ChIP-seq: reads were aligned to the mm10 mouse genome using bowtie1 v1.2.0 allowing one mismatch per read.
For ChIP-seq: peaks were called using MACS2 v2.1.1.20160309 by comparing ChIP samples with corresponding input chromatin libraries.
For ChIP-seq: SAMtools v1.3.1 was used to generate BAM files for all mapped reads with duplicate reads removed.
For ChIP-seq: BigWig files for input-subtracted normalized ChIP-seq signal were generated using bamcompare v2.5.3 from deepTools.
For RNA-seq: for each sample, raw reads from two sequencing lanes were filtered for quality, trimmed to 40bp, and merged.
For RNA-seq: expression levels of all transcripts were estimated using kallisto v0.43.0 with sequence-bias correction. We used the set of transcripts in the basic GENCODE M15 annotation (Ensembl 90).
For RNA-seq: expression levels for protein-coding transcripts on reference chromosomes (mm10) were extracted, summed by gene, and re-normalized to transcript-per-million (TPM) units.
For RNA-seq: normalized counts per gene were obtained using DESeq2 v1.18.1 with default parameters. The estimated count values from kallisto were supplied to DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files represent normalized input-subtracted ChIP signal. Input and ChIP libraries were normalized using RPKM.
Supplementary_files_format_and_content: bed files represent the summits of the "peaks" of heighted read density that were identified in each ChIP sample compared to its corresponding input chromatin sample.
Supplementary_files_format_and_content: kallisto abundance files for RNA-seq represent estimated abundances for every transcript.
Supplementary_files_format_and_content: TPM value files include normalized expression values per gene for all protein-coding transcripts on reference chromosomes (mm10).
Supplementary_files_format_and_content: DESeq normalized count files represent the normalized counts obtained from supplying the estimated count values from kallisto to DESeq2.

Submission date

Jun 18, 2018

Last update date

Feb 26, 2019

Contact name

Mina L Kojima

Organization name

Whitehead Institute

Lab

Page Lab

Street address

455 Main Street