GSM3375184: RAG1A-acrIIA4-1:3-fig6A-rep2; Homo sapiens; OTHER
DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA All the biotinylated primers are placed within 100-bp from the cut site. In general, PEM-seq can be divided into 3 steps: primer-extension, bridge adapter (with RMB on it) ligation and PCR amplification for Illumina sequencing. For primer extension, Bst polymerase 3.0 (NEB) was used to perform primer extension: 65°C 10min, 80°C 5min. Beads-cleaned products were incubated at 95°C for 5 min and quickly put on the ice for 5min for DNA denaturation. Biotinylated PCR products were enriched by Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher). In the bridge adapter ligation step, ligation reaction was performed in 15% PEG8000 (Sigma) at room temperature overnight. Finally, ligation products underwent nested PCR (Taq, Transgene, China) with I5 and I7 sequence primers for 16 cycles and another PCR (Fastpfu, Transgene, China) tagged with Illumina P5 and P7 sequence. All the PEM-seq libraries were sequenced by Hiseq with 150bp ×2.
GEO Accession
GSM3375184