GSM4610275: ChIP-seq_SCC1_priming; Homo sapiens; ChIP-Seq
Beads were re-suspended in 30μL of ChIPmentation reaction buffer (10 mM Tris-HCl pH 8.0; 5mM MgCl2; 1μL TDE1 (Tn5 enzyme) (Nextera DNA Library Prep Kit, Illumina)). In parallel 1μL of input was diluted in 29μL of ChIPmentation reaction buffer. The samples were incubated in 37° C for 10 minutes followed by washing twice in ice-cold LiCl Buffer and TE Buffer. Next, the beads were suspended in 200µl of Elution Buffer (50mM Tris-HCl pH 7.5; 1% SDS; 10mM EDTA; protease inhibitors (Roche)). In parallel the input reaction was mixed with 170μL of the Elution Buffer. All samples were incubated at 65°C overnight for decrosslinking followed by treatment with PureLink RNaseA (Thermo Fisher Scientific) and Proteinase K. The DNA was purified with MinElute PCR Purification Kit (Qiagen) (10μL elution). For preparation of sequencing libraries the purified DNA was amplified using Q5 Hot start DNA polymerase using indexing primers described in (Buenrostro et al., 2013). Initial amplification was performed: 98°C 30 seconds; [98°C 10 seconds; 63°C 30 seconds; 72°C 1 minute]x 5 cycles followed by library quantification with qPCR: 95°C 3 minutes; [95°C 10 seconds; 58°C 30 seconds; 72°C 1 minute] and additional PCR amplification (conditions as for the initial 5 cycles). The required number of additional cycles was calculated from the qPCR data by determining the number of cycles to reach 50% of the maximum signal. After library amplification and indexing, DNA was purified and size selected with AMPure XP magnetic beads (Beckman Coulter) according to manufacturer's protocol. After purification the libraries concentration was determined by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) according to manufacturer's protocol. The fragment size was estimated by DNA ScreenTape (Agilent) and the final library quality was measured with KAPA Library quantification kit (Roche) according to manufacturer's protocol. For sequencing, multiplexed libraries were diluted to 2nM concentration (calculated based the on the KAPA Library quantification kit).
GEO Accession
GSM4610275