GSM6323431: 3m-Control3; Mus musculus; RNA-Seq
Isolated retinae from Astro-KO mice were lysed, and total RNA was isolated using the RNeasy Lipid Tissue kit (Qiagen). The sample was assessed using the RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA) and 2100 Bioanalyzer (Agilent). Total RNA samples > 200 ng with an RNA integrity number > 8.0 were sequenced. The sequencing library was prepared using the TruSeq® Stranded mRNA library prep kit (Illumina, San Diego, CA, USA). Poly-A RNA was purified using poly-T oligo-attached magnetic beads. The eluted mRNA was then fragmented by incubation with the first-strand buffer at 94 °C for 8 min. First-strand cDNA was synthesized by mixing the cleaved RNA fragments, Superscript II reverse transcriptase, and random primers and incubating in a thermal cycler (25 °C for 10 min; 42 °C for 15 min; 70 °C for 15 min; hold at 4 °C). The cDNA was labelled using a cDNA plate barcode label reagent. The sample was then mixed with DNA polymerase I, RNase H, and the barcode label reagents and incubated at 16 °C for 10 min to synthesize second-strand cDNA and digest the template RNA. The cDNA was purified with AMPure XP beads, the 3' ends were adenylated using an A-tailing mix, and the mixture was incubated in a thermal cycler (37 °C for 30 min; 70 °C for 5 min; hold at 4 °C). The adapters were ligated to the cDNA using a ligation mix, and the mixture was incubated at 20 °C for 15 min. After several rounds of PCR amplification, the enriched DNA fragments were purified by AMPure XP beads. The quality and quantity of the library were then validated with the Agilent 2100 Bioanalyzer. Bulk RNA sequencing was performed with an Illumina NovaSeq 6000 following the manufacturer's procedures.