GSM9063652: Tumor_NStat3_KO_02; Mus musculus; RNA-Seq
Total RNA were extracted by using RNeasy Micro Kit (50) (Qiagen, 74004). TANs were FACS sorted into 350 µL Buffer RLT and vortexed properly to lyse cells. 350 µL of 70% ethanol added to the lysate, and mixed well by pipetting. The sample transferred to RNeasy MinElute spin columns in a 2 ml collection tubes and centrifuged for 15 s at 8000 x g. The flow-through were discarded. 350 µl Buffer RW1 added to the RNeasy MinElute spin columns and centrifuged  for 15 s at 8000 x g. The flow-through were discarded. 10 µl DNase I stock solution added to 70 µl Buffer RDD. The DNase I incubation mix (80 µl) directly added to the RNeasy MinElute spin column membrane and incubated for 15 min RT. 350 µl Buffer RW1 was added to the RNeasy MinElute spin column and centrifuged for 15 s at 8000 x g. The collection tubes were discarded. The RNeasy MinElute spin columns were placed in new 2 ml collection tubes. 500 µl Buffer RPE were added to the spin columns and centrifuged for 15 s at 8000 x g. The flow-through were discarded. 500 µl of 80% ethanol were added to the RNeasy MinElute spin columns and centrifuged for 2 min at 8000 x g. The collection tubes were discarded. The RNeasy MinElute spin columns were placed in new 2 ml collection tubes. The spin columns were centrifuged at full speed for 5 min with open lid to dry the membrane. The flow-through and collection tubes were discarded. The RNeasy MinElute spin columns were placed in new 1.5 ml collection tubes. 20 µl RNase-free water were directly added to the center of the spin column membrane and centrifuged for 1 min at full speed to elute the RNA. RNA extracts were kept at -80°C until processed further with bulkseq RNA libraries for RNA-seq were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's protocol.