GSM7428303: sgGgnbp2_cyto_d1_rep2_CW_41; Mus musculus; RNA-Seq
2x106 cells were taken for total RNA extraction as previously described using Bioline RNA isolation kit. Remaining 10x106 cells were used for fractionation using the NE-PER kit (Thermo) following the manufacturer's instructions with modifications for RNA extraction as follows. Briefly, cells were washed 2 x with PBS which was thoroughly removed on the second wash and the pellets were fractionated following instructions with 5 x protease, 1x phosphatase and 1 unit/100uL RNase inhibitor added to CERI and NER buffers. Prior to nuclear extraction, the nuclear pellet was gently washed 2 x with cold PBS to limit cross-over of cytoplasmic fraction. Fractions were prepared for RNA extraction. Liquid extraction protocol was followed from Bioline RNA isolation kit using 600uL of RLY buffer and 6uL of DTT for both the cytoplasmic and nuclear fractions. For fractionation experiment, libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina due to a lack of availability of the Kapa kit (NEB #E7760, E7765).