title: GSM8656364: DarG silenced (ChIP/ADPrSeq) affinity purified for ADP-ribose, replicate 1; Mycobacterium tuberculosis variant bovis; ChIP-Seq
library_construction_protocol: Cultures were centrifuged and resuspended in TE pH8.0.  An equal volume of methanol:chloroform (2:1) was added, and cultures were rocked for 5 minutes by hand.  The bacilli were centrifuged and the pellet dried, then resuspended in TE pH8.0 and distributed into Lysing Matrix B tubes (MP Biomedicals).  An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added, and the bacilli were disrupted using a FastPrep homogeniser.   The mixture was centrifuged to pellet the debris and to separate the phases.  The upper aqueous phase was subjected to a further extraction with phenol:chloroform:isoamyl alcohol, and two extractions with chloroform.  gDNA was precipitated with 0.1 volumes of 3M sodium acetate pH5.2, and 1 volume of propan-2-ol, pelleted by centrifugation, washed with 70% ethanol, resuspended in TE pH8.0 and frozen at -20°C. 10µg aliquots of gDNA were digested in a total volume of 250 µL with 20U of Alu1 enzyme (NEB) and 1µg RNAse (DNase free; Roche) in 1x CutSmart buffer, overnight at 37°C.Fragmented DNA was purified using the Genomic DNA Clean and Concentrator Kit – 10 (Zymed), following the manufacturer's protocol. Libraries were prepared using the TruSeq DNA Library Preparation kit following to manufacture's instructions