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Sample GSM1864826

Query DataSets for GSM1864826

Status

Public on Sep 01, 2015

Title

E13Sertoli_input_2

Sample type

SRA

Source name

E13.5 preSertoli cells

Organism

Mus musculus

Characteristics

strain: C57BL/6J x CD-1 (F1)
tissue: fetal testis
developmental stage: E13.5
antibody: none, input

Extracted molecule

genomic DNA

Extraction protocol

FACS purified preSertoli cells were pelleted, resuspended in 360 µl of PBS and cross-linked with 10 µl of 37% formaldehyde at room temperature for 10 minutes. Cross-linking was stopped by addition of 46.3 µl of 1M glycine for 5 minutes at room temperature. Cells were then pelleted, supernatant was removed and stored at -80oC. 1M cells were pooled from multiple sorts washed twice in 500 µl of PBS with protease inhibitors. Cells were resuspended in 500 µl of lysis buffer (50mM Tris-HCL, 10mM EDTA, 1% SDS and protease inhibitors) and sonicated with a Branson 450 Sonicator (output power of 3, duty cycle of 30% for 16 cycles of 30 seconds with 1 minute rest time between sonications). Bead-antibody complexes were prepared by incubating 30 µl of dynabeads (Protein A; Life Technologies 10002D) with 2.5 µg antibody (Rabbit-anti-H3K27ac; Abcam ab4729). The sonicated lysate was spun down at 4oC for 10 minutes at 10 krpm with 40 µl of the supernatant set aside as input and 200 µl transferred to tubes containing pre-incubated bead-antibody complexes. We added 700 µl of ChIP Dilution Buffer (CDB) (1 % Triton X-100 (Sigma T8787), 2mM EDTA, 150mM NaCl, 20mM Tris (pH=8.0)) with protease inhibitors to IP tubes, and incubated overnight at 4oC. 160 µl of CDB and 8 µl of 5M NaCl were added to the input tube which was incubated at 65oC overnight. The following day, IP tubes were washed as follows: Once with Wash Buffer 1 (50mM Tris HCl, 1mM EDTA, 150 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.1% Sodium deoxycholate), twice with Wash Buffer 2 (50mM Tris HCl, 1mM EDTA, 500 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.1% Sodium deoxycholate), once with Wash Buffer 3 (10mM Tris HCl, 1mM EDTA, 1% NP-40, 1% Sodium deoxycholate, 250mM LiCl), twice with Wash Buffer 4 (50mM Tris HCl, 1mM EDTA, 500 mM LiCl, 1% NP-40, 0.7% Sodium deoxycholate), twice with TE buffer (pH=8.0). All washes were done in 1 ml, with added protease inhibitors, at 4oC for 5 minutes. Solutions for wash buffers were modified from the protocols posted on the Epigenomics Roadmap website. DNA-protein complexes were eluted twice from the beads with 100 µl elution buffer (100mM sodium bicarbonate, 1% SDS, 8mM NaOH). 8 µl of NaCl was added to the eluates, as well as the input tube, and incubated at 65oC overnight. Samples were treated with 1 µl RNase-cocktail (Life Technologies AM2286) for 30 minutes at 37oC, then 4 µl of 0.5M EDTA, 8 µl of 1M Tris and 1 µl of Proteinase K was added for 60 minutes at 45oC. Finally, DNA was purified using PCR purification columns (Qiagen; 28104).
For library preparation for sequencing, DNA was concentrated using a vacuum centrifuge to ~10 µl. 10 µl of IP DNA and 1 µl of input DNA was used in the library preparation using the Rubicon ThruPLEX FD kit according to the manufacturer’s protocol. Size selection of smaller size amplified DNA was done with SPRI beads (Agencourt AMPure XP A63880) at 0.6x concentration. Sequencing was performed at Duke's Genome Sequencing and Analysis core facility on the Illumina HiSeq2000/2500.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Base calls were performed using CASAVA version 1.8.2.
ChIP-seq reads were aligned with Bowtie (Langmead et al., 2009) with only uniquely aligning reads used for future processing. Peaks were called with SICER (Zang et al., 2009) with enrichment called for the histone modifications using the input track as the control. The species variable was set to mm9, redundancy threshold to 2, window size to 200, fragment size to 150, effective genome fraction to 0.7, gap size to 600 and FDR to 0.01.
Genome_build: mm9
Supplementary_files_format_and_content: .txt file contains bed file of peaks across both replicates

Submission date

Aug 31, 2015

Last update date

May 15, 2019

Contact name

Danielle Maatouk

E-mail(s)

dmaatouk@gmail.com

Organization name

Northwestern University

Street address

303 E. Superior St., Lurie 7-250