title: GSM6701362: Ythdf2 cKO, r2, Cage #2; Mus musculus; RNA-Seq
library_construction_protocol: Tumors were harvested and minced into small pieces. Tumor tissue was submerged in RPMI with 10 U/mL DNase I (Thermo Fisher Scientific, EN0521) and 1 mg/ml collagenase IV (Gibco, 17104019) and digested at 37 °C for 1 hour. The suspension was filtered through a 70-mm cell strainer. Red blood cells were lysed with ACK Lysing Buffer (Gibco, A1049201) and resuspended in FACS buffer (DPBS with 2% FBS). Tumor-infiltrating lymphocytes were enriched by CD4 (TIL) Microbeads (Miltenyi Biotec, 130-116-475).  Enriched lymphocytes were stained DAPI, CD45, and CD4. Live CD45+CD4+GFP+ Treg cells were sorted at 4 °C into Trizol (Invitrogen). RNAs were isolated by chloroform and precipitated by isopropanol following the manufacturer's protocol. Genomic DNA was removed by DNase I and purified by RNA Clean & Concentrator. RNA libraries were constructed with the SMARTer Stranded Total RNA-Seq Kit v2 (Takara, 634412).