GSM6935791: H226 SWTX143 3; Homo sapiens; RNA-Seq
Cells were washed with cold PBS before RNA extraction using RNeasy Kit (Qiagen, # 74004) following the manufacturer recommendations First poly-A containing mRNA molecules purification was performed using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments underwent the addition of a single 'A' base and subsequent ligation of the adapter. The products were then purified and enriched through PCR amplification. We then quantified the PCR yield by Qubit and pooled the samples together to make a single strand DNA circle (ssDNA circle), which generated the final library. DNA nanoballs (DNBs) were generated with the ssDNA circle by rolling circle replication (RCR) to enlarge the fluorescent signals during the sequencing process. The DNBs were loaded into the patterned nanoarrays, and pair-end reads of 100bp were read on the BGISEQ-500 platform for the following data analysis