title: RNA-seq of mus musculus: 4h_rep3
design: Total RNA was isolated using the RNeasy Mini Kit (Qiagen). Briefly, harvest a maximum of 1 x 107 cells, as a cell pellet. Add the appropriate volume of Buffer RLT 600 _l; Centrifuge the lysate for 3 min at maximum speed; Carefully remove the supernatant by pipetting, and then add 1 volume of 70% ethanol to the lysate, and mix well by pipetting; Transfer up to 700 _l of the sample, including any precipitate, to an RNeasy Mini spin column placed in a 2 ml collection tube; Close the lid, and centrifuge for 15 s, at 8000g, Discard the flow-through; Add 700 _l Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15 s, at 8000g, Discard the flow-through; Add 500 _l Buffer RPE to the RNeasy spin column, Close the lid, and centrifuge for 15 s at 8000 g, Discard the flow-through; Add 500 _l Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2 min at 8000g; Place the RNeasy spin column in a new 1.5 ml collection tube. Add 50 _l RNase-free water directly to the spin column membrane, Close the lid, and centrifuge for 1 min at 8000g to elute the RNA. RNA samples were treated with Dnase I (Promega) at a concentration of 1 unit/g of total RNA. Total mRNA was purified from 6 g of total RNA using oligo (dT) magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature and the cleaved RNA fragments were used for first strand cDNA synthesis using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNaseH. These cDNA fragments then went through an end repair process and ligation of adapters. These products were purified and enriched by PCR to construct a final cDNA library, which was sequenced using the BGISEQ500 sequencing platform with a sequencing class of PE 111.