GSM8441859: T cell KO1, KO2, KO3 pool, scRNAseq; Mus musculus; RNA-Seq
PI- Vb5.1-5.2+ CD4+ splenic OTII T cells were sorted into 96 well plates using a FACS Melody(18) and then lysed in a buffer containing dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5'-anchor sequence. A reverse transcription reaction, which adds 2–5 untemplated nucleotides to the cDNA 3' end, was then performed. A template-switching oligo carrying 2 riboguanosines and a modified guanosine was added to produce an LNA as the last base at the 3' end. After the first-strand reaction, the cDNA was amplified using 25 cycles. Next, Tn5 tagmentation was used to construct sequencing libraries from the amplified cDNA (Nextera XT DNA Library Preparation Kit, Illumina). The sequencing was carried out using NovaSeq6000 equipment (150PE, Illumina).