GSM8275205: KO3; Mus musculus; RNA-Seq
About 1× 104 ILC3s (Lin−CD45.2intThy1.2hi) were sorted from the large intestine of Trmt61a△Rorc and Trmt61afl/fl mice separately and were lysed in Trizol (Invitrogen). The total RNA was extracted. Biological duplicates were generated for each group. The total RNA sample was digested by DNaseІ (NEB), and purified by oligo-dT beads (Dynabeads mRNA purification kit, Invitrogen), then poly(A) containing mRNA were fragmented into 130 bp with First-strand buffer. First-strand cDNA was generated by N6 primer, First Strand Master Mix and Super Script II reverse transcription (Invitrogen) (reaction condition: 25 °C for 10 min; 42 °C for 40 min; 70 °C for 15 min). Then add Second Strand Master Mix to synthesize the second-strand cDNA (16 °C for 1 h). Purified the cDNA with Ampure XP Beads (AGENCOURT), then combine with End Repair Mix, incubate at 20 °C for 30 min. Purified and add A-Tailing Mix, incubate at 37 °C for 30 min. Then combine the Adenylate 3′ ends DNA, Adapter and Ligation Mix, incubate the ligate reaction at 20 °C for 20 min. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products were purified with Ampure XP Beads (AGENCOURT). The Qualified libraries will amplify on cBot to generate the cluster on the flowcell (TruSeq PE Cluster Kit V3–cBot–HS, Illumina). The amplified flowcell will be sequenced pair end on the HiSeq 2000 System (TruSeq SBS KIT-HS V3, Illumina), read length 50.