GSM5831619: CLP_K3; Mus musculus; RNA-Seq
Total RNA was extracted from fluorescent-activated cell sorting-purified cells by TRIzol. BGI-Tech (Shenzhen, China) constructed the mRNA library and performed sequencing. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. The mRNA was then 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. A single 'A' base was added to the cDNA fragments, followed by subsequent ligation of adapter. The products were then purified and enriched with PCR amplification to generate single strand DNA circles (ssDNA circles). 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 single-end read of 50 bp were read through on the BGISEQ-500 platform for the following data analysis study.