Direct zol rna miniprep w zymo spin iic columns

Melanomas were sequenced from snap-frozen tumors (Yale and CSU cohorts) or low passage cell cultures (<4) as previously described^{2 (link),4 (link)} (Supplementary Data 1, 8). DNA from melanoma cells and freshly frozen tumors was extracted with the DNeasy purification kit (Qiagen Inc., Valencia, CA). High melanin content was removed with OneStep™ PCR Inhibitor Removal Kit (Zymo Research Corporation, Irvine, CA). Direct-zol™ RNA MiniPrep w/ Zymo-Spin™ IIC Columns (Zymo catalog no. D4019) were used to extract RNA from tumors, and the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen, catalog no. 15055-50) was used to extract RNA from peripheral blood mononuclear cells (PBMCs) and melanoma cells.

RNA was extracted from human biopsies, mouse colon and ileum and HT-29 cells using TRIzol reagent (Invitrogen) and Direct-zol™ RNA MiniPrep w/ Zymo-Spin™ IIC Columns (Zymo Research, Irvine, CA, USA). RNA samples extracted from DSS-treated colon tissue were subjected to a LiCl precipitation according the procedure of Viennois E. et al. [33 (link)]. Briefly, 0.1 vol of 8 M LiCl (Sigma-Aldrich, St. Louis, MO, USA) in RNase-free water was added to the RNA elution and the samples were precipitated at 4 °C for 2 h. Samples were then centrifuged at 14,000 × g for 30 min at 4 °C and the supernatant carefully decanted. The resulting pellet was gently resuspended in RNase-free water and the LiCl precipitation was repeated. As LiCl is a known PCR inhibitor, the samples were precipitated once more for 30 min at −20 °C in 0.1 vol of 3 M sodium acetate (NaOAc), pH 5.2 and 2 volumes of 100% ethanol. Samples were centrifuged at 14,000 × g for 30 min and 4 °C, and the supernatant decanted. The resulting pellet was washed twice with 70% ethanol to remove excess salt and resuspended in RNase-free water.