GSM5034274: IL-22+ T cells _WT_rep4; Mus musculus; RNA-Seq
5000 IL-22+ (CD4+IL-22(tdtomato)+) cells were FACS sorted directly into TLC Buffer (Qiagen) with 1% beta-mercaptoethanol. For the preparation of libraries, cell lysates were thawed and RNA was purified with 2.2x RNAClean SPRI beads (Beckman Coulter Genomics) without final elution. The RNA captured beads were air-dried and processed immediately for RNA secondary structure denaturation (72˚C for three minutes) and cDNA synthesis. SMART-seq2 was performed on the resultant samples following the published protocol with minor modifications in the reverse transcription (RT) step. A 15 µL reaction mix was used for subsequent PCR and performed 10 cycles for cDNA amplification. The amplified cDNA from this reaction was purified with 0.8x Ampure SPRI beads (Beckman Coulter Genomics) and eluted in 21ul TE buffer. We used 0.2ng cDNA and one-eighth of the standard Illumina NexteraXT (Illumina FC-131-1096) reaction volume to perform both the tagmentation and PCR indexing steps. Uniquely indexed libraries were pooled and sequenced with NextSeq 500 high output V2 75 cycle kits (Illumina FC-404-2005) and 38 × 38 paired-end reads on the NextSeq 500 instrument. Reads were aligned to the mouse mm10 transcriptome using Bowtie, and expression abundance TPM estimates were obtained using RSEM.  RNA libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM5034274