title: GSM9464053: Single cell CD4+CD44hiTomato+ isolated from PR8 infected mice 9 d.p.i, lung, medLN and spleen, exp 4; Mus musculus; RNA-Seq
library_construction_protocol: Single-cell suspensions were prepared from spleen, lung, lymph node, or intestinal tissues by mechanical dissociation or enzymatic digestion using collagenase and DNase I, followed by Percoll gradient centrifugation where applicable. CD4⁺ T cells were enriched by negative magnetic selection. Fate-mapped CD44⁺Tomato⁺ T cells were isolated by fluorescence-activated cell sorting (FACS). For scRNA-seq, sorted cells were immediately processed for droplet encapsulation. For plate-based TCR sequencing, single cells were index-sorted into lysis buffer and stored at −80 °C prior to reverse transcription. Single-cell RNA-seq libraries were generated using the 10x Genomics Chromium Single Cell 3′ platform according to the manufacturer's instructions. Feature barcode (TotalSeq-C hashtag) libraries were prepared in parallel. Plate-based single-cell TCRβ libraries were generated by RT-PCR amplification from sorted single cells, followed by multiplexed barcoding and Illumina TruSeq Nano library preparation. Libraries were sequenced on Illumina NextSeq 500 (scRNA-seq) or MiSeq (TCR sequencing) instruments.