GSM5448166: WT_2; Homo sapiens; RNA-Seq
Total RNA from OPM2 cells was extracted via RNeasy Plus Mini Kit (QIAGEN). RNA concentration, RNI value, 28S/18S and the fragment length distribution were detected via Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit). The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T ooligoattached magnetic beads. Following purification, theThe first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR amplification. We then quantified the PCR yield by Qubit and pooled samples together to make a single strand DNA circle (ssDNA circle), which gave the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals at the sequencing process. The DNBs were loaded into the patterned nanoarrays and pair-end reads of 100 bp were read through on the DNBseq platform for the following data analysis study. For this step, the DNBseq platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.