alias: GSM3473433
title: GSM3473433: IP_G7_72h_H3K4ME3_2; Hordeum vulgare; ChIP-Seq
library_construction_protocol: Harvesting 3g young leaves per IP, then using a final concentration at 1% formaldehyde to crosslink cells. After stop crosslink with glycine, shearing the chromatin to 100-300 bp with sonication method. With the function of protein G beads, adding specific antibody to pull down the interest protein. Antibodies used in this study are: anti-H3K4me3 (9571 CST), anti-H3K27me3 (9733 CST), anti-IgG (2729 Lot 7 CST). Then digest the protein with proteinase K, DNA which is binding to the interest protein is harvested. Libraries were prepared according to Illumina's instructions accompanying the  NEXTflex® Rapid DNA-Seq Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
identifiers.namespace: GEO
identifiers.id: GSM3473433