2 protocols
normalization data transformation protocol
Pulldown reads were aligned to the hg19 genome assembly using BWA version 0.5.9-r16 Peaks were called using MACS version 1.4.2 with default parameter Genome_build: hg19 Supplementary_files_format_and_content: MACS-generated peaks file.
nucleic acid library construction protocol
Purified genomic DNA should be sonicated into short fragments (200~300 bp) by Covaris DNA shearing with microTUBEs according to manufacturer’s instructions. 5hmC DNA capture was performed using Hydroxymethyl Collect kit. Briefly, 5-hmC labeling reaction were performed in 75-ul solution containing 50mM HEPES buffer(PH7.9), 250 mM MgCl2, 100 µM UDP-6-N3-Glu,and 80 U βGT. The reactions were incubated for 2h at 37℃.After the reaction, The DNA substrates were purified and exchange buffer in H20 via Bio-rad micro bio spin according to the manufacturer’s instructions. The click chemistry was performed with the addition of 150µM biotin into the DNA solution and incubated 2h at 37℃.The DNA samples were then purified by Invitrogen Dynabeads MyOneTM Streptavidin C1 according to manufacturer’s instructions. 5-hmC enriched genomic DNA libraries were generated following the Illumina protocol for “Preparing Samples for CHIP sequencing of DNA”.we used 1ng 5-hmC enriched DNA to initiate the protocol. DNA fragments of 150~300bp were gel purified after the adapter ligation step. PCR-amplified DNA libraries were quantified on an Agilent 2100 Bio analyzer and using quantity PCR.We performed 100bp single end sequencing on Illumina Hiseq2000 to get 5-hmC-enriched DNA fragment sequence.