GSM5290738: gRNA3_2_ANTI_FLAG; Mus musculus; ChIP-Seq
150mm tissue culture dishes containing 90% confluent NIH-3T3 cells from each experimental condition were cross-linked and immunoprecipitated with the Active Motif ChIP-IT High Sensitivity Kit according to manufacturers instructions and using Sigma M2 ANTI-FLAG antibody (F1804). Samples were sonicated on the Bioruptor (Diagenode) in 1.5 mL Eppendorf tubes at high power for three 10-minute cycles of 30 seconds on and 30 seconds off, replacing warmed water with ice-cold water and minimal ice between each cycle. 1% of sonicated chromatin was purified according to ChIP-IT Kit protocol and quantified by NanoDrop. 30 µg input chromatin was used for immunoprecipitation. Fragmented DNA from 12 samples (three replicates each of gRNAscr anti-FLAG, gRNA3 anti-FLAG, gRNAscr input, and gRNA3 input) was quantified using 2100 Bioanalyzer (Agilent Technologies). Libraries were generated robotically with the maximum volume provided by the client of fragmented DNA (range 100-300 bp) using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), as per the manufacturer's recommendations. Adapters and PCR primers were purchased from Integrated DNA Technologies (IDT). Size selection was carried out using SparQ beads (Qiagen) prior to PCR amplification (12 cycles). Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems). Average size fragment was determined using a LabChip GX (PerkinElmer) instrument.