title: NextSeq 500 sequencing; RNA-seq of HCT116 KRAS G13D and HCT116 KRAS WT cell lines with control or CFIm25 knockdown
design: RNA-seq of HCT116 KRAS G13D and HCT116 KRAS WT cell lines with control or CFIm25 knockdown
library_construction_protocol: HCT116 KRAS G13D, KRAS WT cells and DLD-1 cells were maintained in McCoy's 5A medium supplemented with 10% FBS. Pancreatic BX PC3, CFPAC I and HPAF II cell lines were grown in DMEM supplemented with 10% FBS. Cell lines were validated by STR profiling.  RNA from human cancer cells was extracted 48h post transfection using the RNeasy Kit from Qiagen according to the manufacturer's instructions.  For sequencing of human cancer cell samples, total RNA was treated with the DNA-free DNA Removal Kit (cat # AM1906; Thermo Fisher Scientific Inc., Waltham, USA) to remove contaminant DNA. DNase-treated total RNA was then quantified using Qubit RNA BR (cat # Q10211, Thermo Fisher Scientific Inc., Waltham, USA) fluorescent chemistry and 1 ng was used to obtain RNA Integrity Number (RIN) using the Bioanalyzer RNA 6000 Pico kit (cat # 5067-1513, Agilent Technologies Inc., Santa Clara, USA). Lowest RIN was 9.1; median RIN score was 9.8. 1000 ng per sample was then processed using the TruSeq Stranded mRNA Library Prep (cat # 20020595; Illumina Inc., San Diego, USA; protocol v. 1000000040498 v00) including PolyA selection, with 8 minutes of fragmentation at 94 °C and 15 cycles of amplification. 1uL top stock of each purified final library was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip (cat # 5067-4626, Agilent Technologies Inc., Santa Clara, USA). The libraries were quantified using the NEBNext Library Quant Kit for Illumina (cat # E7630L, New England Biolabs, Ipswich, USA) and were pooled at equimolar ratios after size-adjustment. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using the NEBNext Library Quant Kit.