Illumina HiSeq 1500 sequencing; Impact of RNA isolation methods for RNA-seq on gene expression. Here RNA was isolated from HEK293T cells using either Columns, magnetic beads with/without proteinase K or magnetic beads without proteinase K but heating. RNA was processed using prime-seq.
Impact of RNA isolation methods for RNA-seq on gene expression. Here RNA was isolated from HEK293T cells using either Columns, magnetic beads with/without proteinase K or magnetic beads without proteinase K but heating. RNA was processed using prime-seq.
Prior to lysis, cells were stained with 1 ug/ml  Trypan Blue per sample for 3 minutes, and counted using a Neubauer counting chamber. Then, the desired number of cells (1,000 or 10,000) was pelleted for  5 min at 200 rcf, resuspended in 50 µL of lysis buffer RLT Plus with 1% ß-mercaptoethanol   Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM media supplemented with 10% FBS and 100 U/ml Penicillin and 100 μg/ml Streptomycin. Cells were grown to 80% confluency, and harvested by trypsinization RNA was isolated using the bead extraction described in the prime-seq protocol RNA extraction using the Zymo Direct-zol RNA MicroPrep Kit The prime-seq protocol was performed as described here: https://www.protocols.io/view/prime-seq-s9veh66