title: GSM1892437: Input - Chromatin sample 2; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin from three independent biological preparations of THY1+ spermatogonia at passages 9-11 were used for ChIP essentially as described previously (Hermann et al., 2008;Hermann and Heckert, 2005). Briefly, cells were fixed by addition of 1% v/v formaldehyde to the culture media at RT°C for 15 min and fixation was quenched with addition of excess glycine (to 125mM). Cells were scraped, pelleted and washed twice with DPBS + 0.5% IGEPAL. Cell pellets were subjected to one freeze-thaw cycle, suspended in lysis buffer (10mM EDTA, 50mM Tris pH 8.1, 1% SDS and 1X protease inhibitor cocktail) and cells were disrupted with a dounce homogenizer. Chromatin was sheared to an average length of 300-500 bp via sonication and quantified by spectrophotometry. An aliquot of chromatin (Input) was treated with RNase, proteinase K and heated to reverse the crosslinks, and then isolated with ethanol precipitation. Aliquots of chromatin (30 µg) were precleared with protein-A or protein-G agarose beads (Invitrogen) and then incubated with 5ug of primary antibodies for PLZF (goat anti-PLZF IgG, sc-11146, Santa Cruz Biotechnology), RNA polymerase II phospho-CTD-Ser2 (mouse anti-POLII-pSer2-CTD, 61083, Active Motif) and SALL4 (rabbit anti-SALL4 IgG, ab29112, Abcam) at 4°C overnight. Protein-A or Protein-G agarose beads were then used to isolate immune complexes from SALL4 and PLZF/POLII IPs, respectively, and then washed, eluted with SDS buffer, and treated with both RNase A and proteinase K. Following overnight incubation at 65°C to reverse crosslinks, ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. To confirm chromatin quality prior to next-generation sequencing, ChIP qPCR was performed for RNA polymerase II pCTD-Ser2 in triplicate reactions with primers against intronic regions of the Actb (+1831) and Gapdh (+2089) genes using SYBR Green Supermix (Bio-Rad; Supplemental Fig. 2A). Quantitative PCR signals were normalized to those obtained from input DNA and calculated as copies of DNA detected per 1000 genome equivalents of input DNA (1000 cells). A genomic region located in a gene desert on Chromosome-6 (Untr6) served as a negative control as described (Franco et al., 2012). For next-generation sequencing, 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 Genome Analyzer II or HiSeq2000 instruments.
attributes.tag: GEO Accession
attributes.value: GSM1892437