GSM9210935: RAW264.7 cells, shTrem2, rep3; Mus musculus; RNA-Seq
Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer' s instructions. Then RNA quality was determined by 5300 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample(OD260/280=1.8~2.2, OD260/230≥2.0, RQN≥ 6.5, 28S:18S≥ 1.0, >1g) was used to construct sequencing library. RNA purification, reverse transcription, library construction and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer's instructions (Illumina, San Diego, CA). The RNA-seq transcriptome librariy was prepared following Illumina® Stranded mRNA Prep Ligation from Illumina (San Diego, CA) using 1μg of total RNA. Shortly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer firstly. Secondly double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by Qubit 4.0, paired-end RNA-seq sequencing library was sequenced with the NovaSeq X plus sequencer (2 × 150bp read length).