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Sample GSM2202696

Query DataSets for GSM2202696

Status

Public on Apr 24, 2017

Title

TSPAN8_High_Input

Sample type

SRA

Source name

P6 ID4-EGFP+ spermatogonia from mouse testes

Organism

Mus musculus

Characteristics

strain: LT-11B6 (C57BL/6)
cell population: TSPAN8-high subpopulation of P6 ID4-EGFP+

Treatment protocol

N/A

Growth protocol

LT-11B6 (Id4-eGfp transgenic mice on C57BL/6 background) from Jon Oatley (Washington State University; Chan et al., 2014 Genes & Development 28: 1351-1362) were bred to produce pup male mice. Testes from postnatal day 6 (P6) pups were used to produce testis cell suspensions that were stained with antibodies against TSPAN8 and subsequently used for FACS. For the "high" subpopulations of ID4-EGFP+ spermatogonia (based on TSPAN8 antibody labeling), the top 1/3 most robustly stained cells were sorted, while for the low subpopulations, the bottom 1/3 most weak positive stained cells were selected.

Extracted molecule

genomic DNA

Extraction protocol

Sorted P6 ID4-EGFP+ spermatogonia and subpopulations defined by antibody labeling for TSPAN8 were used for Reduced-representation bisulfite sequencing (RRBS) analysis of methylome or ChIP-seq analysis of post-translational modifications of histones (H3K4me3, H3K27me3 and H3K27Ac). For RRBS, snap-frozen cell pellets from five independent sorts of TSPAN8-High and TSPAN8-Low ID4-EGFP+ spermatogonia were used for genomic DNA isolation and RRBS was performed using the Methyl-MidiSeq service by Zymo Research (Irvine, CA). For ChIP-seq, snap-frozen cell pellets from eight independent sorts of the TSPAN8-High and TSPAN8-Low subpopulations were subjected to low-input ChIP-seq service by Active Motif (Carlsbad, CA) using proprietary approaches. Briefly, native (unfixed) chromatin was isolated, quantified, and 9000 (H3K4me3) to 34000 (H3K27me3 and H3K27Ac) cell equivalents of chromatin (6.6pg/cell) were used for each ChIP reaction using antibodies against H3K4me3 (Active Motif 39159), H3K27Ac (Active Motif 39133) or H3K27me3 (Millipore, 07-449). ChIP'd genomic DNA or genomic DNA from residual chromatin were subsequently used for sequencing.
For RRBS, libraries were prepared from 300 ng of genomic DNA digested with the BfaI, MseI, and MspI restriction enzymes and the fragments produced were ligated to pre-annealed adapters containing 5’-methyl-cytosine instead of cytosine. Adapter-ligated fragments were filled in and 3’-terminal-A extended, then purified using the Zymo Research (ZR) DNA Clean & Concentrator – 5 kit (Cat#: D4003). Bisulfite treatment of the fragments was done using the EZ DNA Methylation – Lightning kit (ZR, Cat#: D5030). PCR was performed and the size and concentration of the fragments were confirmed on the Agilent 2200 TapeStation, then sequenced on the Illumina Hiseq 2500 with PE50 parameters. For ChIP-seq, ChIP and input samples were prepared for amplification by converting overhangs into phosphorylated blunt ends, 3’ adenylation, ligation of Illumina adaptors and library size selection (175-225 bp) on an agarose gel. Adaptor-ligated libraries were then amplified for 18 cycles and resulting DNAs were purified, quantified, and tested by qPCR to assess quality of amplification reactions. Amplified DNA libraries subjected to Illumina sequencing with HiSeq2000 instrument (SE50).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Data processing

For RRBS, Sequence reads from bisulfite-treated EpiQuest libraries were identified using standard Illumina base-calling software and then analyzed using a Zymo Research proprietary analysis pipeline implemented in Python (using the mm10 genome annotation). Residual cytosines (Cs) in each read were first converted to thymines (Ts), with each such conversion noted for subsequent analysis. A reference sequence database was constructed from the 50-bp ends of each computationally predicted MspI-TaqI fragment in the 40–350 bp size range. All Cs in each fragment end were then converted to Ts; the converted reads were aligned to the converted reference by Bowtie. The number of mismatches in the induced alignment was then counted between the unconverted read and reference, ignoring cases in which a T in the unconverted read is matched to a C in the unconverted reference. For a given read, the best alignment was kept. If there was more than one best alignment, the read was discarded as non-unique. The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Fisher’s exact test or t-test was performed for each CpG site which has at least 5 reads covered. Also, promoter, gene body and CpG island annotations were added for each CpG. Genome positions were converted to mm9 for data presentation and final processing by liftover from mm10 to mm9.
For ChIP-seq, reads from each sample were aligned to mouse genome (mm9) using ELAND (Illumina), sequence aligns (75 bases) were then 3’ extended in silico to a length of 200bp and assigned to 32bp bins along the genome, normalized to the same number of unique reads (~16M) and peaks were called using the SICER algorithm (Zang et al., Bioinformatics 25, 1952-1958, 2009) at a cutoff of FDR≤1E-10 and a Gap parameter of 600 bp (which merges peaks located within 600 bp of each other into a single “island”).
Genome_build: mm10 (RRBS) or mm9 (ChIP-seq)
Supplementary_files_format_and_content: RRBS results are shown in CpG_result_table.txt.gz, which is a matrix table listing all detected CpG dinucleotides and methylation status (%) in both samples and significance calls.
Supplementary_files_format_and_content: ChIP-seq results are found in TSPAN8_ChIP_Intervals.xlsx, which is an excel spreadsheet with three matrix tables (three tabs), each listing all of the intervals (peaks) found in each sample type. Each tab/table lists results from one histone modification (H3K4me3, H3K27Ac and H3K27me3, respectively).

Submission date

Jun 16, 2016

Last update date

May 15, 2019

Contact name

Brian P. Hermann

E-mail(s)

Brian.Hermann@utsa.edu

Phone

210-458-8047

Organization name

University of Texas at San Antonio