GSM7976219: CD8 T cells with low 2-NBDG uptake; Mus musculus; RNA-Seq
RNA was extracted with the RNeasy Microkit (Qiagen, Catalog: 74004). cDNA libraries were prepared from poly A RNA (mRNA) using the Smart-Seq2 single-cell protocol with some modifications. Briefly, reverse transcription was done with SuperScript™ II reverse transcriptase (Invitrogen, Catalog: 18064014), oligo-dT30VN (1 µM; 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′), template-switching oligonucleotides (1 µM) and betaine (1 M). Template switching was done using a locked nucleic acid (LNA) without the purification step before preamplification PCR to obtain an increased cDNA yield. cDNA concentration was measured with the Qubit dsDNA High Sensitivity assay (Invitrogen, Catalog: Q32851) and analyzed with Agilent Bioanalyzer or Fragment analyzer High Sensitivity assay (Agilent, Catalog: 5067‐4626 or DNF‐474) to check the size distribution profile. cDNA libraries were prepared using NEBNext® Ultra DNA Library Prep for Illumina® kit (NEB, catalog: E7370) according to the manufacturer's protocol. Briefly, 5 ng of cDNA were fragmented at a range size of 200‐500bp using Covaris S2, then subjected to end repair and addition of “A” bases to 3′ ends, ligation of adapters and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (Beckman Coulter, catalog: A63882,). Library amplification was performed by PCR using NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1, Catalog E7335), (Index Primers Set 2, Catalog: E7500), (Index Primers Set 3, Catalog E7710) or/and (Index Primers Set 4, Catalog: E7730). Final libraries were analyzed with Agilent Bioanalyzer or Fragment analyzer High Sensitivity assay to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems, Catalog: KK4835,) prior to amplification with Illumina's cBot. Libraries were sequenced 1 x 50 + 8 bp on Illumina's HiSeq2500.