GSM4274350: WT-2 [ChIP-seq]; Mycolicibacterium smegmatis MC2 155; ChIP-Seq
The cells were fixed with 1% formaldehyde for 30 min, the reaction was quenched with 150 mM glycine for 15 min, and then the cells were washed three times with cold PBS (54,000 rpm, 5 min, 4 °C) and frozen at -80 °C. To prepare lysates, pellets were resuspended in FA-1 buffer (HEPES-KOH at 50 mM [pH 7.5], NaCl at 140 mM, EDTA at 1 mM, Triton X-100 at 1%, and protease inhibitor cocktail [Sigma-Aldrich]), disintegrated with silica beads (0.1 mm) for 45 min, and sonicated on ice using ten cycles of a 10-s pulse followed by a 50-s pause. The obtained lysates were centrifuged (5 min, 12,000 rpm, 4 °C) and frozen at -80 °C in 5% glycerol. For immunoprecipitation, 200 μg of total protein was incubated on a rotary shaker (Thermo Fisher Scientific, China Co., Ltd.) for four hours at 4 °C with a 15-μl packed-gel volume of anti-FLAG M2 magnetic beads (Sigma-Aldrich) and then washed twice with FA-1 buffer. Samples were processed in a final volume of 0.5 ml in two biological replicates, with input DNA controls (200 μg of total protein alone) included for each replicate. Samples were washed using a magnetic separator with sequential applications of FA-1 buffer, FA-2 buffer (HEPES-KOH at 50 mM [pH 7.5], NaCl at 500 mM, EDTA at 1 mM, Triton X-100 at 1%, and protease inhibitor cocktail), and Tris-EDTA buffer solution (TE) (Tris-HCl at 10 mM [pH 8.0], EDTA at 1 mM). Immunoprecipitated samples were de-cross-linked overnight in TE containing 1% SDS at 65 °C and then digested with proteinase K (final concentration, 0.05 mg/ml) for 1.5 h at 55 °C. The immunoprecipitated DNA was extracted using Cycle-Pure Kit (OMEGA Bio-tek, Inc.). The library of DNA fragments was prepared using a QIAseq Ultralow Input library kit (Qiagen). Briefly, the protocol includes DNA end-repair, sequencing adapter ligation, cleanup, and PCR amplification. At the end of the procedure, quantification and quality evaluations were done using a NanoPhotometer® spectrophotometer (IMPLEN), a Qubit® 2.0 Fluorometer (Life Technologies), and a 2100 Bioanalyzer (Agilent). Second-generation sequencing was performed using a HiSeq 1500 sequencing platform (Illumina).
GEO Accession
GSM4274350