title: Illumina HiSeq 2000 sequencing; RNA-seq on human colon cancer cell line GP5d with siRNA against CTNNB1, MAX and MXD1 against control siRNA
design: RNA-seq on human colon cancer cell line GP5d with siRNA against CTNNB1, MAX and MXD1 against control siRNA
library_construction_protocol: GP5d cells were trypsinized and washed with PBS. GP5d human colon adenocarcinoma cells (ECACC cat no. 95090715) were cultured in DMEM supplemented with antibiotics and 10% FBS under standard cell culture conditions. Reverse transfections were performed with 50 μl of HiPerfect (Qiagen, Cat. No. 142349463) in 750 μl of Opti-MEM (Invitrogen), according to manufacturer's general instructions using 40 nM final concentration for siRNA (non-targeting siCtrl, ThermoFisher, Cat. No. D-001206-13-20; siCTNNB1, Dharmacon, Cat. No. M-003482-00-0005; siMAX, Santa Cruz, Cat. No. sc-38079, and siMXD1, Ambion, Cat. No. s8390) in 100 mm dishes. Cells (25 ×10^5 of cells), added on top of the transfection complex, were cultured for 72 hr before harvesting. RNA was extracted with RNeasy Mini Kit (Qiagen, Cat No. 74104). RNA was extracted with RNeasy Mini Kit (Qiagen, Cat No. 74104) For RNA-sequencing, NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, NEB #E7420S) was used for library preparation. However, due to technical problems, the libraries are not strand-specific.