title: GSM8893623: HEK293T, dCas13-NES-eNAT10 and NT gRNA transfection, isotype control IP, rep1; Homo sapiens; RIP-Seq
library_name: GSM8893623
library_construction_protocol: Total RNA was isolated and purified using a PureLink RNA Mini Kit (Invitrogen). For acRIP-seq, one day before immunoprecipitation, Protein G Dynabeads (Invitrogen) was mixed with antibody and incubated overnight at 4℃ with rotation. 1.5 ug of rabbit anti-ac4C (Abcam, ab252215) and 1.5 ug of rabbit isotype IgG (CST, #3900) was used for each sample. In both cases (anti-ac4C and isotype controls), 35 ug of isolated total RNA was mixed with antibody-conjugated beads. Samples were incubated for 4 hours at 4℃ with rotation. After incubation, beads were washed three times with RIP buffer, and bound RNA was eluted using lysis buffer provided in the PureLink RNA Mini Kit, supplemented with 1% beta-mercaptoethanol (Sigma). Eluted RNA was purified using an RNA Clean & Concentrator kit (Zymo Research). For ac4C-seq, total RNA was split into two reactions: the first half (10 ug) was incubated with 10 ul of 1 M NaCNBH3 (Sigma) and 10 ul of 1 M HCl (Sigma), and the reaction volume was adjusted to 100 ul with nuclease-free distilled water; the second half (NaCNBH3-untreated controls) was the same except that NaCNBH3 was replaced with 10 ul of distilled water. Reactions were carried out for 20 minutes at room temperature, then were terminated by addition of 30 ul of Tris-HCl pH 8.0 (LPS solution). RNA was precipitated with 3 M sodium acetate (Invitrogen) and 70% ethanol. For fractionation-seq, transfected cells grown in 6-well cell culture plate were harvested and resuspended with 200 µl lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630 in DPBS). Cells were lysed on ice for exactly 2 minutes and the lysis was terminated by adding 500 ul resuspension buffer (1% bovine serum albumin, 0.2 U/ul murine RNase inhibitor in DPBS). Samples were centrifuged at 500g for 10 minutes at 4℃, and supernatant was carefully harvested for cytoplasmic fraction. Pallets were washed two more times with 500 ul resuspension buffer, and resulting pallet was harvested for nuclear fraction. Cytoplasmic fraction and nuclear fraction were subjected to RNA isolation Sequencing library was constructed from RNA samples using SureSelect RNA Direct kit (Agilent).