GSM3356865: SUDHL4_H3K4me1_si; Homo sapiens; ChIP-Seq
Briefly, SUDHL4 were fixed, lysed, and sonicated to generate fragments less than 400 bp. Sonicated lysates were incubated with antibodies against LSD1 (abcam 17721), BCL6 (Santa Cruz N3, sc-858), or H3K4me1 (abcam, ab8895), and after increasing stringency washes, immunocomplexes were recovered and DNA was isolated. H3K4me1 ChIPs also contained Drosophila spike-in chromatin control (750ng per ChIP, Active Motif, 53083) added during precipitation along with Drosophila antibody (2ug, Active Motif, 61686) per manufacturer's recommendation for proper signal normalization. ChIP-seq libraries were prepared using the Illumina ChIP-seq Library preparation Kit following the manufacturer's instructions with minor modifications. Briefly, 10ng of purified ChIP DNA (quantified using Qubit 2.0 fluorometer, Invitrogen) was end repaired by conversion of overhangs to phosphorylated blunt ends. A' bases were added to the 30 ends of the DNA fragments and Illumina adapters (1:30 dilution) were ligated to the ends of ChIP fragments. After adaptor ligation, DNA was separated by electrophoresis and size selected by isolating a gel band of 250 ± 25bp. Size selected fragments were PCR amplified for 15 cycles using Illumina genomic DNA primers 1.1 and 1.2 with the following program (30 s at 98oC, 15cycles of 10 at 98oC, 30 s at 65oC, 30 s at 72oC and 5min extension at 75oC). Q-PCR was repeated to confirm retention of relative enrichment.