title: GSM6701355: WT, Input, r1, RIP-seq; Mus musculus; RIP-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 into lysis buffer (50 mM HEPES, 150 mM KCl, 2 mM EDTA, 0.5% NP40, 0.5 mM DTT, protease inhibitor (Roche), and SUPERase·In RNase Inhibitor (Thermo Fisher Scientific, AM2696) and placed on ice for 30 minutes. 20 mL Protein A Dynabeads (Thermo Fisher Scientific, 10008D) were washed with lysis buffer and incubated with 2 mg anti-YTHDF2 for 30 minutes at 4 °C. Beads were washed twice with lysis buffer. Cell lysis was centrifuged at 20,000 × g for 15 minutes. 10% of the lysis was stored in Trizol as the input, and the rest was added to bead-antibody conjugates to rotate at 4 °C for 4 hours. After incubation, beads were washed with NT2 buffer (50mM HEPES, 200mM NaCl, 2mM EDTA, 0.05% NP40, 0.5mM DTT, protease inhibitor, and RNase inhibitor) 6 times and resuspended in Trizol. 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).