title: RNA-seq of 293T cell : CRISPRa treatment
design: The Ribo-off rRNA Depletion Kit (Vazyme, cat. no. N406) was used to remove ribosomal RNA (rRNA) in total RNA. Briefly, 1g total RNA was diluted with ddH2O to a final volume of 11l. 1 uL rRNA probe and 3 uL probe buffer were added to the RNA sample and mixed thoroughly. The mixture was heated to 95C for 2min and then cooled to 22C at a rate of 0.1  per second and incubated at 22C for 5min. Next, RNase H and RNase H buffer were added to the above products and mixed, which were immediately transferred to ice after incubation at 37 C for 30 min. The remaining rRNA probes in the samples were then digested with DNase I at 37 C. The samples and VAHTS RNA Clean Beads (Vazyme, cat. no. N412) were incubated on rice for 15min to allow the non-ribo RNA to bind to the beads. The non-ribo RNA was then separated and purified using a magnetic rack. 18.5l of 1 x Frag/Prime Buffer was added to elute the non-ribo RNA from the beads. The eluted samples were heated for 8min at 94C in a thermocycler, followed by a hold at 4C. Then the cDNA library was constructed according to the VAHTS universal V8 RNA-seq library prep kit for Illumina (Vazyme, cat. no. NR605).
identifiers.namespace: SUB14899629
identifiers.id: bulkRNAseq_293T_miniCRISPRa