GSM8509658: NC, Immune cell, rep2; Mus musculus; RNA-Seq
Total RNA was extracted from 100 mg of mouse retina using TRIzol solution (15596018, Invitrogen, CA, USA), according to the manufacturer's specifications. Contaminated genomic DNA was removed from 10 μg of total RNA by the incubation with 20 units of RNase-free DNase I (New England Biolabs) and 4 units of RNase inhibitor (New England Biolabs) in DEPC-treated water. The reaction mixture was incubated for 1 h at 37 °C and then for 10 min at 60 °C. The RNA samples quantified spectrophotometrically at 260 nm and all RNA extracts had an OD260:OD280 between 1.8 and 2.0, demonstrating that RNA was suitable for subsequent analyses. Oligo‐dT (6110A, Takara) was used as the primer in the first step of cDNA synthesis. Total RNA (1 μg) was combined with 1 μl of oligo dT, and H2O and then preheated at 70 °C for 10 min to denature the secondary structures of RNA. The mix was then cooled off quickly to 4 °C, subsequent to which 10 mM DTT, 2 μl of 10X reverse-transcriptase buffer, and 200 units of reverse transcriptase (18064022, Invitrogen, CA, USA) was added to make a total volume of 20 μl. The reverse-transcriptase mixture was incubated at 40 °C for 60 min, after which it was stopped by heating at 94 °C for 20 sec. The construction of RNA-sequencing library was performed using TruSeq RNA Sample preparation kit v2 (Illumina, Inc., San Diego, CA, cat. no. RS-122–2002). In briefly, 100 ng of total RNA from each sample was exposed to poly-T oligo-attached magnetic beads to isolate poly-A mRNA following mRNA fragmentation. The cleaved RNA fragments were constructed onto a double-stranded cDNA.  After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. Then, the double-stranded library was purified using AMPure XP beads (A63881, Beckman coulter, CA, USA) to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library is purified from PCR components. Libraries were analyzed by an Agilent 2100 Bioanalyzer using the High-Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA). Then, the cDNA libraries were used for paired-end 75 sequencing using an Illumina NextSeq 500 (Illumina, Inc., San Diego, CA).