GSM3895899: HiC-Cast-rep2; Mus musculus; Hi-C
Splenic and lymph node CD4+ T cells from all mice were prepared by MACS purification with CD4 microbeads (Miltenyi Biotec), and the purity was usually 90%–95%. Naive T cells were obtained by surface staining with anti-CD4, anti-CD62L, anti-CD44, and anti-CD25. The CD4+ CD62L+ CD44CD25 population was isolated by flow-cytometry cell sorting with a Mo-Flo cell sorter (Dako, Carpinteria, CA). The three enzyme Hi-C (3e Hi-C) was performed according to our previous studies. About 1,000,000 CD4+ naïve cells were stained with Biotin anti-CD45.2 antibody, bound to biotin binder (Dynabeads Biotin Binder, Cat#11047, Invitrogen) and cross-linked with 1% formaldehyde for 10 mins. Biotin binders were blocked by 0.5mM biotin solution. Cells were lysed and subsequently digested with 20 Units CviQ I (NEB), and 20 Units CviA II (NEB) at 25 C for 20 minutes, then 20 Units Bfa I (NEB) at 37 C for 20 minutes. The reaction was stopped by washing the cells twice with 600 mL wash buffer (10mM NaCl, 1mM EDTA, 0.1% triton-100). DNA ends were marked by biotin-14-dATP with Klenow (large) for 1hr at 37 C. Blunt-end DNA fragments were ligated with T4 DNA Ligase overnight at 16 C. DNA was then reverse cross-linked and purified by phenol/chloroform extraction. Biotin was removed from unligated DNA-ends by T4 DNA polymerase for 2hrs at 12 C. DNA was purified by phenol/chloroform and sheared to 300-500 bp by sonication followed by DNA-end repair and addition of ''A''. Biotin labeled DNA was pull-downed by streptavidin beads followed by Illumina adaptor ligation and PCR amplification. DNA fragments of 300 to 700 bp were isolated 2% agarose gel and sequenced by paired-end sequencing on Illumina Hiseq 2500. ChIP-Seq experiments were performed as described previously. In brief, five millions of CD4 Naïve T cells were fixed for 10 min with 1% formadehyde in complete medium, sonicated and chromatin immunoprecipitation was performed with antibodies against CTCF (07-729, Millipore), RNA Polymerase II (ab5408, Abcam), H3K4me3 (ab8580, Abcam) and H3K4me2 (ab32356, Abcam). The DNA was then ligated with the ''Y''-shaped Illumina adaptor and amplified for 18 cycles using indexing primers as described. PCR products between 160-300bp were isolated on 2% E-gel for sequencing on Illumina Hiseq2500. Total RNA was purified with an miRNeasy micro kit (217084; Qiagen) and a DNase set (79254; Qiagen) for the option of on-column digestion with DNase. 10 ng purified total RNA was used for amplification of cDNA with the Ovation RNA-Seq System V2 (7102-08; NuGEN Technologies). 200 ng cDNA was sonicated in a Diagenode Bioruptor (level M, for a total of 30 min of 20s on and 20s off) to size range of 100–400bp. Indexed libraries were prepared with a Multiplexing Sample Preparation Oligonucleotide Kit (1005709; Illumina) and an End-It DNA End-repair Kit (ER81050; Epcentre) according to the user's manual (Epcentre) and sample preparation guide (Illumina).