alias: GSM2051454
title: GSM2051454: c-Myc_ChIP-Seq; Mus musculus; ChIP-Seq
library_construction_protocol: Tissue were cut into small pieces, washed with cold PBS, and fixed with 1% formaldehyde for 20 min at room temperature; fixation was stopped by addition of glycine (0.125 M, final concentration) with an additional incubation of 5 min. Cells were collected by centrifugation and washed twice with cold PBS. After centrifugation, the cells were pelleted and lysed for 30 min in 700ml of SDS buffer (50mM Tris pH 8.1, 100mM NaCl, 5mM EDTA, 0.5% SDS) supplemented with protease inhibitors (Roche #11873580001). Chromatin was disrupted using a Covaris instrument during 40 min (20% duty cycle; 10% intensity; 200 cycle), yielding DNA fragments with a bulk size of 300–500 bp. Samples were centrifuged to pellet cell debris. The amount of chromatin isolated was quantified in a Nanodrop; an aliquot of this material was used as input for sequencing. To each sample (500mg of chromatin for ChIP or 1mg for ChIP-seq) Triton dilution buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) was added to a total of 1 mL and the sample was pre-cleared during 2h with a mix of protein G/A (previously blocked with 5% BSA) at 4 ºC. We used 10μg of anti-c-Myc antibody (N262, Santa Cruz) pre-bound overnight to 100μl of G protein in PBS with 0.5% BSA. Beads were added to samples and incubated overnight at 4 °C in a rotating platform. After washing and elution, crosslinks were reversed as described by (Aparicio et al. 2005). This included successive washes in 1 mL of Mixed Micelle Buffer (20mM Tris at pH 8.1,150 mM NaCl, 5mM EDTA, 5% w/v sucrose, 1% Triton X-100, and 0.2% SDS), Buffer 500 (50mM HEPES at pH 7.5, 0.1% w/v deoxycholic acid, 1% Triton X-100, 500mM NaCl, and 1mM EDTA), LiCl Detergent Wash Buffer (10mM Tris at pH 8.0, 0.5% deoxycholic acid, 0.5% NP-40, 250mM LiCl, and 1mM EDTA), and TE (pH 7.5). Crosslinks were reversed by incubation overnight at 65 °C in Elution Buffer (1% SDS, 0.1M NaHCO3). The eluted DNA was treated with proteinase K solution (10mM EDTA, 40mM Tris-HCl pH 6.5 and 40mg/ml proteinase K) and purified by phenol-chloroform extraction; the DNA pellet was resuspended in 40ml. DNA (20ng) was quantified by fluorometry, resolved by electrophoresis, and fractions of 50-250 bp were extracted. Input samples correspond to balanced blends of inputs from selected samples. Fractions were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters following Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated libraries were amplified by limited-cycle PCR with Illumina PE primers (12 cycles). The resulting purified DNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents following manufacturer's protocols.
identifiers.namespace: GEO
identifiers.id: GSM2051454