GSM8442160: HepG2 H3K4me3 replicate 2; Homo sapiens; ChIP-Seq
Fixed cells were subjected to sonication following the NEXSON procedure. Briefly, nuclei were isolated by sonication using a Q Sonica Sonicator with settings of 15 seconds On, 30 seconds Off for 8 cycles at 20% power (2 minutes total sonication time).  Nuclei were centrifuged at 1250 RCF and resuspended in CHIP Buffer.  DNA was sheared using sonicator settings of 30 seconds On, 30 seconds Off at 80% power for 4 cycles of 5 minutes each (20min. total sonication time). Samples were centrifuged at 18,000 RCF and supernatant was collected and tested on an agarose gel to check for size, phenol extracted, precipitated, and resuspended.  ChIP was carried out with 300ng sheared DNA and the Chip-IT High Sensitivity Kit (Active Motif, 53040) according to kit instructions with the following antibodies from Active Motif: H3K27ac (39685), H3K4me1 (39297), H3K4me3 (61379), and anti-mouse IgG (53017). Libraries were generated from the resulting material using the ThruPLEX Tag-seq Library Kit (Takara, 030520), PCR amplified for 10-16 cycles then purified and size selected using AMPure XP Beads.  Completed libraries were checked for quality and quantity on a BioAnalyzer 2100 and Qubit respectively.  Libraries passing QC were pooled at 1nM and sequenced on a Novaseq 6000 using an S1 flow cell (51bp paired end read length, dual indexed).