alias: BB2_GC
title: H3K4me2_ChIPseq_input_control[BB2_GC]
design: epididymides and vas deferens were excised; the vas deferens was immediately placed in a 37C phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) and incubated at 37C for five minutes to facilitate spermatozoa swim-out. Approximately 2.5mL of sperm cell suspension was collected from epididymides 60 and vas deferens pooled for each rat. The total sperm concentration was determined using a hemocytometer. Following sperm collection, each sample was split with 4 million sperm for a genomic control library and 10 million sperm for immunoprecipitation. Genomic control samples were subjected to a modified QIAamp DNA Micro Kit extraction protocol. The immunoprecipitated sample was made using a modified nChIP-seq protocol by doubling the time for MNase digestion (to a total of 10 minutes), using 30 U enzyme per two million sperm, and using 5 ug of ChIP-seq validated H3K4me2 antibody. Libraries were prepared from four paired genomic control and immunoprecipitated samples using NuGen's Ovation Ultralow Library System V2 average size and raw concentration of each library was determined using 2 Fragment Analyzer (AATI) with a High Sensitivity NGS Fragment Analysis kit (cat# DNF 486-0500). Subsequently, each library was diluted into three different concentrations and quantified by qPCR in 85 triplicates using the KAPA quantification Kit. Samples were then multiplexed across two lanes. Genewiz (www.genewiz.com) performed sequencing using 50bp single end reads on an Illumina HiSeq2500 platform.
identifiers.namespace: SUB4576742
identifiers.id: BB2_GC