title: GSM1678942: SREB-3xHA B ChIP; Blastomyces dermatitidis ATCC 26199; ChIP-Seq
library_construction_protocol: B. dermatitidis ATCC 26199 and SREB-3xHA cells were grown in liquid HMM containing 10 μM iron sulfate (FeSO4) at 37oC overnight and treated with 1% formaldehyde for 30 minutes.  Formaldehyde cross-linking was quenched with 0.125 M glycine for 5 minutes. Cells were washed with ice-cold 1x phosphate buffered saline (PBS) containing a protease inhibitor cocktail (PIC; Sigma Aldrich), flash frozen in liquid nitrogen, ground to a fine powder using a a mortar and pestle, and resuspended in FA lysis buffer with PIC plus PMSF.  Chromatin was sheared using probe sonication (Misonix S-4000) for a total of 25 minutes of "on time".  Each round of sonication was divided into 15 seconds (amplitude 30) x 4 with intervening rest periods on ice lasting 30 seconds.  Prior to each round of sonication, cells were chilled for approximately 10 seconds in a dry-ice ethanol bath to prevent overheating.  From each sample, 50 μl was saved as the "input" sample and 50 μl was analyzed for efficacy of chromatin shearing by 0.8% agarose gel electrophoresis.  For chromatin immunoprecipitation (ChIP), protein A agarose beads (without salmon sperm DNA) were prepared according to the manufacturers instructions (Millipore) and resuspended in FA lysis buffer containing PIC and PMSF.  Sonicated chromatin (300 μg measured by nanodrop spectophotometer) was pre-cleared for 1 hour at 4oC prior to adding 10 μl ChIP-grade anti-HA tag antibody (Abcam, catalogue # ab9110) along with freshly prepared beads.  Following overnight incubation at 4oC, samples were washed with with buffer #1 (FA lysis buffer), once with buffer #2 (FA lysis buffer with 0.5 M NaCl), once with buffer #3 (10 mM Tris pH8, 0.25M LiCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate), and three times with Tris-EDTA pH 8.  All washes were 3 minutes at 4oC with orbital rotation.  ChIP'd samples were treated with proteinase K for 2 hours at 45oC and then 65oC overnight. Input samples were treated with proteinase K at 55oC x 4 hours then 65oC overnight.  DNA was isolated and purified according to the protocol in Mukhopadhyay et al. [Nat Protocol 2008;3:698-70] and resuspended in Tris-HCl pH 8.  The DNA from 3 technical replicates of ChIP'd or input samples were pooled for each biological replicate of ATCC 26199 and SREB-3xHA.  There were 2 biological replicates for ATCC 26199 and SREB-3xHA. Immunoprecipitated and input DNA were submitted to the University of Wisconsin Biotechnology Center in Madison, WI for sequencing using Illumina HiSeq 2000 platform.
attributes.tag: GEO Accession
attributes.value: GSM1678942