title: GSM4572199: anti-LOS2 antibody; Arabidopsis thaliana; ChIP-Seq
library_construction_protocol: The 7-d-old wild-type seedlings grown on 1/2 MS medium were fixed in 1% formaldehyde for 15 min at room temperature under a vacuum. Fixed tissues were ground into a fine powder in liquid nitrogen and the nuclei were isolated. The chromatin released from the nuclei was sonicated into 100–500 bp fragments. One third of the resulting DNA fragments were used as an input, with the remaining two thirds used for immunoprecipitation with or without an anti-LOS2 antibody, respectively. The immunoprecipitated DNA fragments were used to perform ChIP-seq experiment." The DNA is combined with End Repair Mix, and incubate at 20 for 30 min. Purify the end-repaired DNA with QIAquick PCR Purification Kit(Qiagen)then add A-Tailing Mix, incubate at 37 for 30 min. Combine the purified Adenylate 3'Ends DNA, Adapter and Ligation Mix, incubate the ligation reaction at 20 for 15 min. Purify the Adapterligated DNA with the QIAquick PCR Purification Kit. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the Adapter-ligated DNA fragments. Then the PCR products are selected (about 100-300bp, including adaptor sequence) by running a 2% agarose gel to recover the target fragments. Purify the gel with QIAquick Gel Extraction kit (QIAGEN). The final library is quantitated in two ways: Determine the average molecule length and sample integrity as well as purity using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and quantify the library by real-time quantitative PCR (QPCR). The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. Library was qualified by Qubit ssDNA kit. The sequencing will be performed with the BGISEQ-500 sequencing system,featuring combinatorial probe-anchor synthesis (cPAS) and DNA Nanoballs (DNB)technology for superior data quality (BGI-Shenzhen, China). The Qualified libraries will amplify on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3–cBot–HSIllumina). And the amplified flowcell will be sequenced pair end on the HiSeq 2000 System (TruSeq SBS KIT-HS V3, Illumina)