GSM5013034: Th17 differentiated CD4 T Cell - eGFP_neg, IL23R Knockout, cell 234; Mus musculus; RNA-Seq
Spleen and lymph nodes were extracted and then a single-cell suspension was prepared by homogenization with a syringe plunger and debris was filtered with a 70um filter. Red blood cells were lysed with ACK lysis buffer from Lonza. CD4+ T cells were enriched using a bead protocol from Miltenyi Biotech (cat #. 130-117-043) and then stained with anti-CD44 (clone IM7), anti-CD62L (clone Mel14), anti-CD4 (clone RM4-5) and anti-CD25 (clone PC61) in preparation for FACS. Naive T cells were sorted based on CD44lowCD62LhiCD4+CD25-. T cells were sorted into 96-well plates containing 5 μl TCL Buffer (QIAGEN) with 1% 2-mercaptoethanol, centrifuged and frozen at −80 °C.  Single-cell sequencing libraries were then created using the Smart-seq2 protocol (Picelli et al., Nature Methods 2014) and carried out as described in Singer et. al, Cell 2016.