GSM9059601: Treg-enriched lung lymphocytes from uninfected chimeric mice, GEX; Mus musculus; RNA-Seq
Lungs were harvested and digested in RPMI 1640 medium containing 5mg/mL collagenase type 1 and 0.25mg/mL DNaseI for 20 minutes at 37 ⁰C, 5% CO2. Lungs were then dissociated mechanically and passed through a 100uM filter, then multiple lungs were combined and enriched for lymphocytes using a 33% Percoll gradient (Cytiva). Samples were then enriched for regulatory T cells using the mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec) and stored in Cryostor (Sigma) in liquid nitrogen prior to analysis. Cell viability was determined, then cells were stained with TotalSeq C anti-mouse antibodies (BioLegend, San Diego, CA) per manufacturer's protocol. Sample loading resulted in ~10,000 cells for analysis. Chromium X was used to process samples. RNA libraries were created with the Chromium Next GEM Single Cell 5' Kit with Feature Barcoding. Chromium Single Cell Human BCR Amplification Kit was used for V(D)J library construction. Agilent Tapestation (Agilent Technologies, Palo Alto, CA) was used to confirm sequencing library integrity, and the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA), as well as qPCR (Applied Biosystems, Carslbad, CA), was used to quantify libraries. The Illumina sequencing platform was used to sequence libraries as recommended by 10X Genomics.