GSM3615364: F4-combined; Homo sapiens; RNA-Seq
RNA was extracted following manufacturer instructions (RNeasy Mini Kit – Qiagen, #74104), with the addition of an extra DNase digestion step to increase the quality of the RNA. This digestion was performed following the method in appendix E: DNase digestion of RNA before RNA cleanup from the RNase-Free DNase Set (Qiagen, #79254). To 87.5 µl of RNA sample was added 10 µl Buffer RDD and 2.5 µl DNase I stock solution (provided by the Moffitt's Molecular Genomics Core). The sample volumes were brought up to 100 µl with RNase-free water and incubated at room temperature for 10 minutes before proceeding with the cleanup protocol of the RNeasy Mini Kit (Qiagen). Also, we performed an ethanol precipitation after RNA extraction. The volume of each sample was brought up to 200 µl and 1/10 volume (20 µl) of 5 M NaCl plus 2.5 volumes (500 µl) of absolute ethanol were added prior to thorough vortex. Samples were centrifuged at 1,000 xg for 10 minutes; the supernatant was discarded, and pellets were washed with 500 µl of freshly diluted 75% ethanol in RNase-free water. Samples were centrifuged again at 1,000 xg for 10 minutes, supernatant was discarded, and samples were left to air-dry. Samples were resuspended in RNase-free water and analyzed by Nanodrop to verify the 260/280 and 260/230 ratio > 1.8 and ~2.0. RNA-Seq library generation was performed following the Encore Complete RNA-Seq Library System protocol (Nugen). The KAPA library amplification kit for Illumina platform (KAPABiosystem) was used as described in the technical data sheet.  Beads were resuspended in 20 µl of elution buffer.