GSM9045573: LENG8-depletion, cytoplasm mRNA-seq, replicate3; Homo sapiens; RNA-Seq
For fragnation analysis, 150 ŒºL of pre-chilled 0.7% NP-40 lysis buffer (10 mM Tris-HCl, 75 mM NaCl, 0.7% NP-40, pH 7.5, supplemented with fresh protease inhibitors) was added. The suspension was pipetted using a 200 ŒºL tip until homogeneous and incubated on ice for 30 seconds. Samples were then centrifuged at 13,300 rpm for 15 seconds to separate the cytoplasmic (supernatant) and nuclear (pellet) fractions. A total of 150 ŒºL of the cytoplasmic supernatant was collected into new tubes for cytoplasmic RNA extraction by TRIzol reagent. The nuclear pellet was washed three times with 1√ó PBS. 1 mL of TRIzol reagent was added to extract nuclear RNAs. For total RNA, total RNA was extracted from cells using TRIzol reagent Polyadenylated RNA was purified using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and then purified mRNA was fragmented at 94¬∞C for 3 minutes using the NEBNext Magnesium RNA Fragmentation Module. For PAS-seq analysis, only the 3'end of mRNA was used for library constrcution.All of library construction was followed illumina's protocol