GSM6126097: CD4 T Cells, Control rep5; Mus musculus; RNA-Seq
The intestinal CD4+ T cells were isolated from the colons of RAG1-/- recipients using a lamina propria dissociation kit (Miltenyi Biotec) as outlined in Pawlak M., DeTomaso, D. et al. 2021. Briefly, intestines were flushed with ice-cold buffer and opened longitudinally with scissors. The intraepithelial lymphocytes (IEL) were removed with buffer containing 1mM DTT. Lamina propria lymphocytes (LPL) were isolated from the intestinal mucosa by digestion at 37°C using a rotator. After digestion, the samples were homogenized using a gentleMACS (Miltenyi Biotec). CD4+ T cells were sorted into 5 μl TCL Buffer (QIAGEN) with 1% 2-mercaptoethanol, centrifuged and frozen at −80 °C. For library construction, samples were thawed briefly at RT and ~20ul of each was transferred to a 96-well plate. The construction of the full-length RNA-seq libraries followed a modified SMART-Seq2 protocol (Picelli et al., 2013). After first strand cDNA synthesis and amplification, the cDNA was purified with a 0.7x single sided SPRI.  Each sample was normalized to 0.20ng/ul and then tagmented and indexed using the Nextera XT DNA library prep kit (Illumina). The individually barcoded libraries were then pooled and a final quality control was done on the BioAnalyzer (Agilent). The pool was normalized to 4nM, denatured using 0.2 N NaOh and 0.2 M Tris-HCL and diluted in HCL to prepare for sequencing. 1300ul of the final diluted pooled library was loaded for sequencing on a NextSeq 500/550. Paired end sequencing was performed using an Illumina 75 cycle Nextseq 500 high output V2 kit in accordance with Illumina protocol (Reads 38 cycles x2, Index 8 cycles x2).