GSM5035565: activated CD4 T-cells, 48 hours after activation, high CD62L [Set1_48_hi_3]; Mus musculus; OTHER
RNA was extracted using TRI-reagent (Sigma-Aldirch), according to standard protocol. tRNA sequencing protocol was adapted from Zheng et al., 2015 (Zheng et al., 2015) with minor modification. Small RNA was isolated using SPRI-beads (Agencourt AMPure XP, Beckman Coulter), using dual side size-selection protocol. First RNA and beads were mixed at 1:1.8 ratio, collection the supernatant. The small RNA was isolated by mixing the clear supernatant with bead at 1:0.8 ratio, with addition of X1.34 isopropanol. Small RNA was treated for modification removal using AlkB wt, and D135S, a kind gift from Prof. Tao Pan. Reverse transcription was done using TGIRT™-III Enzyme, with the indicated primers. 3' adaptor was ligated to the cDNA using T4 ligase (NEB). The cDNA was purified using Dynabeads myOne SILANE (life Technologies) after each step.  The library was amplified using NEBNext PCR mix and cleaned using SPRI-beads. Samples were pooled and sequenced using NextSeq illumina. primers: Reverse-transcription DNA:5'-CACGACGCTCTTCCGATCTT  -3',  Reverse-transcription RNA:5'-rArGrArUrCrGrGrArArGrArGrCrGrUrCrGrUrG-3',  3' adaptor:5'-  CGTCATAGATCGGAAGAGCACA-3'.
GEO Accession
GSM5035565