title: GSM3754041: RPB1_BF_ChIP; Trypanosoma brucei brucei; ChIP-Seq
library_construction_protocol: T. brucei BS cultures (TREU927 strain) were harvested at 1 x 10E6 parasites per ml and fixed with 1% formaldehyde (5 x 10E7 total parasites per IP). Pellets were resuspended 0.5 ml of lysis buffer, sonicated to shear the chromatin (~300pb in length), diluted 1:5 in ChIP dilution buffer and pre-cleared with Sepharose® 4B beads (Sigma©). Pre-cleared chromatin was incubated overnight at 4 ºC with 10 μg of affinity purified anti-RPB1 antiserum. Next, protein G Sepharose® (Sigma©) was added and incubated for 2h at 4 ºC. Beads were then washed and immunoprecipitated DNA fragments recovered in 200 ul elution buffer. After crosslink reversal, DNA was extracted with phenol:chloroform, ethanol precipitated and resuspended in 50 μl of miliQ water. Four different immunoprecipitates were pulled, concentrated to a final volume of 10 μl using the MinElute® PCR Purification Kit (Qiagen©) and quantified with Qubit® 3.0 fluorometer (ThermoFisher Scientific©) using the QubitTM dsDNA HS Assay Kit. Libraries for NGS were generated with NEBNext® UltraTM II DNA Library preparation kit and indexed with NEBNext® Multiplex Oligos (Illumina®). Paired-end sequencing was run in NextSeqTM 500 device using NextSeqTM 500/550 High Output Kit v2.5 (75 cycles).
attributes.tag: GEO Accession
attributes.value: GSM3754041