GSM5017652: Lsr2-2-Input; Mycolicibacterium smegmatis; ChIP-Seq
The cells were fixed with 0.01% formaldehyde for 30 min, the reaction was quenched with glycine, and washed three times with cold PBS. To prepare lysates, pellets were disintegrated with silica beads and sonicated. The obtained lysates were centrifuged (5 min, 12,000 rpm, 4°C) and frozen. For immunoprecipitation, 700 µg of the total protein was incubated on a rotary shaker for 4 h at 4°C with a 20 µl packed-gel volume of anti-FLAG M2 magnetic beads (Sigma-Aldrich) and then washed. Samples were processed in a final volume of 0.5 ml in two biological replicates, with input DNA controls (70 µg of total protein alone) included for each replicate. Immunoprecipitated samples were de-cross-linked and then digested with proteinase K (final concentration, 0.05 mg/ml). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with absolute ethanol. 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 Nanodrop spectrophotometer (Thermo Scientific), a Quantus fluorimeter (Promega), and a 2100 Bioanalyzer (Agilent). Second-generation sequencing was performed using a HiSeq 1500 sequencing platform (Illumina).