title: GSM5662531: gl2-5_1; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: The 10-day old Arabidopsis seedlings of each genotype were harvested and crosslinked with 1% formaldehyde for 10 min in a vacuum chamber. Glycine was added to a final concentration of 0.125 M, and the reaction was terminated by incubation for 5 min under vacuum. Chromatin was fragmented using a Covaris 220 focused ultrasonicator (Duty Cycle: 10%; Intensity: 5; Cycles per Burst: 200) to produce <500 bp fragments. ChIP experiment was performed with a ChIP-grade anti-GFP antibody as previously described (Khosla et al., Plant Cell 2014; doi: 10.1105/tpc.113.120360). Immunoprecipitated DNA was prepared for sequencing. The ChIP-Seq was performed using the Illumina HiSeq 2500 Sequencing System at the University of Kansas Medical Center – Genomics Core (Kansas City, KS). Fragmented input and immunoprecipitated chromatin (20ng) was used to initiate the TruSeq ChIP Sample Prep Kit library preparation protocol (Illumina cat#IP-202-1012). The fragmented chromatin underwent end repair and 3' adenylation prior to illumina indexed adapter ligation. Library sizing (target size – 320bp) was performed using the Sage Pippin Prep automated size selection system. PCR amplification of the sized library fraction was performed using Illumina adaptor specific priming with final library purification using KAPA Pure magnetic bead purification (KAPA cat#KK8002).  Following Agilent TapeStation 4200 QC for sizing of the library preparation and library quantification using the Roche Lightcycler96 with Roche FastStart Essential Green Master qPCR kit (Roche cat#06402712001 ) and KAPA Library Quant (Illumina) DNA Standards (KAPA cat#KK4903), the CHiP-Seq libraries were adjusted to a 2.125nM concentration and pooled for multiplexed sequencing.