title: GSM2584577: Input ChIP-Seq; Schizosaccharomyces pombe; ChIP-Seq
library_construction_protocol: 100mL cultures grown to between 0.5–0.8 OD600 were crosslinked with 1% formaldehyde (Sigma) for 15 minutes, and then quenched for 5 minutes with 125mM glycine (Sigma). Crosslinked cells were isolated by centrifugation and washed twice with 4°C TBS. Pellets were aliquoted into 50 OD600 units of cells, flash frozen in liquid nitrogen and stored at -80°C. Aliquots were thawed on ice and resuspended in 1 mL ChIP lysis buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 1x EDTA-free Complete protease inhibitor (PI; Roche) Protease inhibitors (2mM PMSF, 20μg/mL leupeptin, 20μg/mL pepstatin, 20μg/mL aprotonin, 20μg/mL antipain). Lysate was generated using a Mini-Beadbeater (Biospec Products) for 8 cycles of 1.5 minutes beadbeating followed by 2 minutes on ice after each cycle. DNA was isolated by centrifugation. DNA was sheared in a Bioruptor waterbath sonicator (Diagenode) with 6 cycles on high (10 min total, 30 sec ON, 1 min OFF). After sonication, cell debris was removed by centrifugation and the supernatant was brought up to 1mL in ChIP lysis buffer. 100μL of the lysate was then removed and saved as an input. 10ug of antibody against M2 FLAG (F3165, Sigma) was added to the remaining 900μL of lysate and this reaction was incubated for 2 hours at 4°C. Prior to addition, 30μL of packed Protein G DYNAbeads (Invitrogen) were washed for 5 minutes each at 4°C: twice with 1mL TBS and then twice in 1mL ChIP lysis buffer, and then resuspended in 100μL ChIP lysis buffer. This 100μL of washed beads was add to the reaction and incubated for 2 hours at 4°C for immunoprecipitation. Beads were then washed for 5 minutes each at 4°C: twice with 1mL ChIP lysis buffer, twice with 1mL high salt ChIP lysis buffer (ChIP lysis buffer with 500mM NaCl), and once with TE at room temperature. Crosslinking was reversed in 1% SDS at 65°C overnight in the presence of Proteinase K (30μL of 20mg/mL). SDS was removed from DNA using NucleoSpin Gel Clean-up Columns with 1250μL NTB (Macherey-Nagel). ChIP DNA libraries were prepared using End-It DNA End Repair Kit followed by A-tailing using Klenow fragment (NEB), ligation to Illumina adaptors using Rapid T4 DNA Ligase (Enzymatics), PCR amplification for 15 cycles, and size selection between 200-350bp
attributes.tag: GEO Accession
attributes.value: GSM2584577