GSM6246203: PolyA-seq_HeLa_MYC.L1-RTm_6; Homo sapiens; OTHER
Genomic DNA was extracted using rapid lysis buffer containing 20 μg/ml Proteinase K and incubation at 56 °C overnight, followed by standard isopropanol extraction, wash in 70% ethanol and resuspension in TE buffer. 50 μg genomic DNA was digested overnight with HaeIII enzyme (recognizes and cuts at 5'-GG/CC-3'), followed by 3'A addition by Klenow polymerase (3′–5′ exo-; New England Biolabs) and adaptor ligation (composed of an upper linker and a lower 3′-modified linker) by T4 DNA Ligase (Promega). To reduce sequencing background, ligation reactions were digested with Xba I to remove the exogenous LINE-1 sequence from plasmid (first blocking). In the first round of PCR, DNA was amplified using Biotin-LEAP, together with adaptor specific reverse primer (AP1) and Phusion polymerase (Thermo Fisher Scientific). 20 PCR cycles were performed in the following conditions: 98 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min. Multiple reactions were performed in generating large-scale libraries. Biotinylated PCR products were captured by Dynabeads MyOne StreptavidinC1 kit (Invitrogen) for 2-3 hours at room temperature with rotation, followed by 2 h digestion with Xba I (second blocking). PCR products were then eluted in 95% formamide/10 mM EDTA and purified by PCR extraction kit (Qiagen). The second round emulsion-PCR (EM-PCR) was performed in an oil-surfactant mixture by nested forward primer (SV40-polyA-F) and nested adaptor specific reverse primer (AP2) using the following conditions: 20 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min. The PCR products was then extracted three times with diethyl ether and DNA was re-purified by PCR extraction kit (Qiagen). The third round of PCR was performed with barcoded forward and reverse primer (10-12 cycles of 98 °C for 20 s, 60 °C for 30 s, and 72 °C for 1 min). PCR products were size fractionated for DNA fragments between 300 bp and 1000 bp on a 1% agarose gel and purified by Gel extraction kit (Qiagen). PolyA-seq