GSM5291027: HelaS3_WT_total_1; Homo sapiens; RNA-Seq
Three individual replicates for each of the two cell lines were collected. A single 15cm dish corresponding to one replicate was harvested at a time. For each replicate, cell media was aspirated and cells washed with ice-cold PBS. No cycloheximide pre-treatment was was performed. After thorough removal of the PBS, the dish was fully immersed in liquid nitrogen and placed on dry ice. For cell lysis, 1ml of 1x Mammalian Lysis Buffer (Illumina) containing 100 μg/ml cycloheximide was added dropwise to the dish which was then placed on wet ice. Cells were then scraped off to the lower portion of the dish and allowed to thaw in the lysis buffer. Lysate was homogenised by pipetting and triturated ten times through a 25-gauge needle. The lysate was then transferred to a DNA LoBind 1.5ml microfuge tube (Eppendorf) and incubated on ice for 5min. The lysate was cleared by centrifugation at 20000g, 4°C for 10 minutes and the supernatant transferred to a fresh microfuge tube. Aliquots of 100 and 200µl were prepared for each replicate, flash-frozen in liquid nitrogen and stored at −80°C until further use. Thereafter, the protocol for TruSeq Ribo Profile Mammalian (Illumina) was followed to generate total RNA and ribosome protected fragment RNA-seq libraries corresponding to 3 individual replicates from each of the two cell lines. TruSeq Ribo Profile Mammalian (Illumina), with the modifications given in "extract protocol" above