title: GSM1173872: ChIP Stat5a replicate; Mus musculus; ChIP-Seq
library_construction_protocol: Chromatin Preparation (ChIP):  EDTA-free protease inhibitors (Roche, France) were added to all washing buffers to a finalconcentration of 1X. Aliquots of 5x107 cells were lysed after resuspension in 2 ml LB1 (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100) for 20 minutes on a rotating wheel at 4°C. Chromatin was collected by centrifugation for 5 minutes at 4°C and 1.350g in a tabletop centrifuge, and washed in 2 ml LB2 (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8,10mM Tris pH 8) on a rotating wheel for 10 minutes at 4°C. Chromatin was collected,resuspended in 2 ml of LB3 (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mMNaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and sonicated using a Misonix 4000(Misonix Inc, USA) sonicator for 16 cycles (30 seconds on, 30 seconds off, amplitude 40) with the tubessubmerged in ice-cold sonication solution (500mM NaCl, 20% ethanol). After the addition ofTriton X-100 to a final concentration of 1%, particulates were collected through centrifugation at20.000g and 4°C for 20 minutes. For batch processing, supernatants were combined, mixedthoroughly and subsequently aliquoted and snap-frozen in liquid nitrogen. Extracts were kept at-80°C until use and 50μl aliquots were taken to serve as input controls.  Inputs were combined with an equal volume of 2x elution buffer (100mM Tris pH 8, 20mM EDTA pH 8, 2% SDS) and incubated overnight in a water bath at 65°C for 13-15 hours. SDS was then diluted by the addition of an equal volume of TE (10mM Tris pH 8, 1mM EDTA pH 8) and RNA was digested by RNase A at a final concentration of 0.2μg/ml at 37°C for 2 hours. Samples were subsequently Proteinase K treated at 55°C for two hours at a final concentration of0.2μg/ml. DNA was purified by two subsequent phenol:chloroform:isoamylalcohol (25:24:1, pH8) extractions and followed by a Qiaquick purification (PCR purification columns, Qiagen,Germany). To ensure greater DNA purity, columns were washed twice with PE buffer and elutedin 50μl H2O. DNA concentration was measured using a Nanodrop 1000 (Thermo Scientific,France) and 1 ul was used to verify sonication efficiencies using High Sensitivity DNA chips on a 2100 Bioanalyzer. ChIP experiments were performed using Dynabeads (Invitrogen, USA) coated with Protein-G. Beads were washed 3x with 1ml and subsequently resuspended in 250μl of blocking solution (0.5% BSA in 1x DPBS). After the addition of the antibody (anti-Stat5a; PA-ST5A from R&D Sysytems), the beads were incubated at 4°C overnight on a rotating wheel. Unbound antibodies were removed through three further washes with 1ml of blocking solution. Beads were resuspended in 100μl of blocking solution, extracts were added and the mix was incubated overnight at 4°C on a rotating wheel. EDTA-free protease inhibitors (Roche) were added to all washing buffers to a final concentration of 1x. Beads were washed 8 times in RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate) and once in TE+ (10mM Tris pH 8, 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatin was recovered from the beads with two subsequent elution steps at 65°C for 15 and 10 minutes in 60μl and 40μl of elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) respectively. The two eluates were combined and incubated at 65°C overnight (13-15 hours) for crosslink reversal. DNA was purified as described for the input.    Sequencing  10 ng of Input and 90 µl of STAT5a ChIP were used for high throughput sequencing. Standard Applied Biosystems Life Technologies protocols were followed for the creation of short-insert bar-coded libraries (insert length ~150 bp) according to the ChIP-seq Kit Guide from the manufacturer (cms_079323.pdf) and library preparation guide (cms_081748.pdf). The library was PCR amplified with 15 cycles. The amplified fragments were verified on a 2100 Bioanalyzer (Agilent, USA) before sequencing on an AB SOLiDTM V4.0 or hq5500XL (Life Technologies) according to the manufacturer’s protocol.