GSM6340306: noTF-condition_ZNF287_siRNA_Rep2; Homo sapiens; RNA-Seq
Total RNA was isolated using the ZR-96 Quick-RNA kit (Zymo Research, R1052), which includes an on-column DNase I treatment step, and eluted in 30 μL RNase-free dH2O. 75-100 ng of isolated RNA was used to prepare RNA-seq libraries for sequencing. All custom primers used in cDNA synthesis are listed below. An oligo-dT primer was used to perform poly-A enrichment and was incubated with the sample for 5 minutes. First-stand cDNA synthesis was performed with Superscript II Reverse Transcriptase (Invitrogen 18064022) and a template switch oligo by incubating for 1.5 hours. Second-strand cDNA synthesis was performed with the KAPA HiFi HotStart Ready Mix (Roche 7958927001) and an IS primer with 3 cycles of PCR amplification. 5ng of cDNA was fragmented using the Tn5 enzyme-adaptor compound. 15 cycles of PCR were then carried out with barcoded primers compatible with the BGISEQ-500. The 300-500bp DNA fragments were selected and purified. The fragments were then heat-denatured and one of the single strands was circularized with DNA ligase to obtain a single strand circular DNA library. The remaining single strand was digested with the exonuclease. The sequencing process was conducted according to the BGISEQ-500 protocol as described (Huang et al. 2017).  Primers used in cDNA prep:  Oligo-dT: 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′   TSO: 5′-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3′  (rG= riboguanosine, +G= locked-nucleic acid)  IS Primer: 5′-AAGCAGTGGTATCAACGCAGAGTAC-3′