GSM8616960: miR-26bKO2; Mus musculus; RNA-Seq
Total RNA was isolated  using Qiazol reagent and miRNeasy mini Kit (217004, QIAGEN, Germany). Agilent 2100 Bio analyzer (Agilent RNA 6000 Nano Kit) was used to do the total RNA sample QC: RNA concentration, RIN value,28S/18S and the fragment length distribution. The first step in the workflow involved 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. The PCR yield was then quantified  by Qubit and samples pooled together to make a single strand DNA circle (ssDNA circle), which gives 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  following data analysis study.  The DNBseq platform combines the DNA nanoball-based nanoarrays and stepwise sequencing using Combinational Probe-Anchor Synthesis Sequencing Method.