GSM9340577: siSpp1_rep2; Mus musculus; RNA-Seq
Total RNA was extracted from bone marrow-derived macrophages using the RNeasy Plus Micro kit (Qiagen) according to the manufacturer's protocol. RNA concentration and integrity were measured using the RiboGreen assay (Thermo Fisher Scientific) and the Agilent TapeStation 4200, respectively. Next, polyadenylated mRNAs were captured using oligo-dT-conjugated magnetic beads (Watchmaker Genomics), followed by immediate fragmentation to 300-400 bp. Stranded libraries were then made by adding dUTP into the second strand with the Watchmaker mRNA Library Prep kit (Watchmaker Genomics) and X-Gen stubby adapters (Integrated DNA Technologies). Libraries were enriched and indexed using 10 cycles of amplification with the Equinox Master Mix (Watchmaker Genomics) and PCR primers which included dual 8 bp index sequences to allow for multiplexing (Integrated DNA Technologies). All library preparation volumes were miniaturized to 1/5 reaction volume, followed by removal of excess PCR reagents through magnetic bead-based cleanup using Aline PCRClean Dx paramagnetic beads. The resulting libraries were validated and quantified with the Agilent TapeStation 4200 and by real-time qPCR and subsequently sequenced on an Illumina NovaSeq X Plus using paired-end, 150 bp reads.