GSM6523928: BMDM co-cultured 4T1 ADAM17 knock-out 2.8, repeat 2; Mus musculus; RNA-Seq
RNA was harvested using Rneasy mini plus kit (Qiagen), including DNase digestion. 200 ng of total RNA was used for the construction of sequencing libraries. The 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.