GSM9844138: M1 cells-shNT control, rep3; Mus musculus; RNA-Seq
Total RNA was extracted using Direct-zol™ RNA Miniprep Kit (Zymo Research), and 600 ng of total RNA was used for mRNA purification. The cDNA libraries were prepared using the Illumina® Stranded mRNA Prep, Ligation kit (Illumina) as per the manufacturer's instructions. Briefly, polyA RNA was purified from 200 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3'– end adenylation, and adaptor ligation, followed by PCR amplification and SPRI bead purification (Beckman Coulter). The unique dual index sequences (IDT® for Illumina® RNA UD Indexes Set A, Ligation, Illumina) were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The libraries were pooled and diluted to 3 nM using 10 mM Tris-HCl, pH 8.5 and then denatured using the Illumina protocol. The denatured libraries were loaded onto an S1 flow cell on an Illumina NovaSeq 6000 (Illumina) and run for 2X53 cycles according to the manufacturer's instructions.