GSM5009389: ilet7-Ctrl_rep3; Mus musculus; RNA-Seq
Cells were dissociated with Trypsin for 5 min, stopped with serum containing media, and depleted with MEF for at least half-an-hour, and total RNA was extracted by TRIzon A total amount of 2 μg RNA per sample was used as input materials for the RNA sample preparation. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Purified mRNA was fragmentated at 94 °C for 15 min by using divalent cations under elevated temperature in NEBNext first strand synthesis reaction buffer (5X). First strand cDNA was synthesized using random primer and ProtoScript II reverse transcriptase in a preheated thermal cycler as follows: 10 min at 25 °C; 15 min at 42 °C; 15 min at 70 °C. Immediately finished, second strand synthesis reaction was performed by using second strand synthesis reaction buffer (10X) and enzyme mix at 16 °C for 1 hr. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, A-tailing and adapter added were implemented. The products were retrieved and PCR was performed for library enrichment. The libraries were sequenced on an Illumina platform.