GSM6266244: AZD4785; Homo sapiens; RNA-Seq
RNA was harvested using Rneasy mini plus kit (Qiagen). 1 ug of total RNA was used for the construction of sequencing libraries. A Nanodrop 1000 spectrophotometer was used to assess RNA purity and concentration. RNA integrity was measured using the RNA 6000 Nano Kit (Agilent) according to the manufacturer's instructions and samples run on the Agilent 2100 Bioanalyzer RNA libraries for RNA-seq were prepared as follows. The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo- attached 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 BGISEQ-500 platform for the following data analysis study. For this step, the BGISEQ-500 platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe- Anchor Synthesis Sequencing Method.