GSM7296063: S47.SLE.BM4.X8; Homo sapiens; RNA-Seq
RNA extraction was done with Qiagen RNeasy plus mini kit (Cat#74134/74136) -RNA was extracted in batched as cell pellets from each group of donors were frozen together in 96 well plates -The plates were removed from the freezer at thawed at 4°C (approx. 1 hour) and stored at 4°C for the day while different wells had RNA extracted -The cell pellets in 125 μl RNAprotect were mixed up and down and moved into 1.5 ml microfuge tube (eppendorf). -Cells were pelleted by centrifuging for 5 mins at 5000 x g. -The SN was then aspirated -RNA was then extracted using the manufacturer's instructions from step 2, Briefly- -The pellets were loosened by flicking -Cells were then disrupted with RLT plus buffer by adding 350 μl RLT plus buffer and vortexing to mix. -Cells were then homogenised by pipetting sample on a QIAshredder column and centrifuging 2 mins at full speed -The lysate was transferred to a gDNA eliminator spin column and centrifuge for 30s at 10,000 x g, collect flow through (can repeat spin if membrane has any remaining liquid) -350 μl 70% ethanol was added and mixed well by pipetting (adjust volume if ethanol if some lysate lost in previous steps) -Sample and any precipitate was transferred to a RNeasy spin column, close the lid gently and centrifuge for 15 sec at 10,000 x g, discard flow through -700 μl buffer RW1 was added to the column, close the lid gently and centrifuge for 15 sec at 10,000 x g, flow through discarded -500 μl buffer RPE was added to the column, close the lid gently and centrifuge for 15 sec at 10,000 x g, flow through discarded -500 μl buffer RPE was added to the column, close the lid gently and centrifuge for 2 mins at 10,000 x g, collection tube and flow through discarded -Column placed in new 2 ml collection tube, lid of spin column opened, spin at full speed for 5 mins, collection tube and flow through discarded -RNeasy spin column placed in a new 1.5 ml collection tube, 30 μl RNase free water (warmed to 55°C) was added directly to the centre of the spin column and incubated for 10 mins, then centrifuged for 1 min at 16,000 x g -An additional 10 μl RNase free water was added, then centrifuged for 1 min at 16,000 x g – approx. 35 μl will be eluted for potential RNAseq -Eluted RNA was immediately stored at -80°C and shipped for sequencing for RNAseq frozen on dry ice RNA libraries were prepared for sequencing using standard Illumina protocols for polyA extraction for samples from stimulated cells.