GSM4174014: Mettl3_Control_mNET_r2; Mus musculus; RIP-Seq
Isolated chromatin was washed in 100 l of 1x Micrococcal Nuclease (MNase) buffer (NEB) and then incubated with MNase (40 u/ml) on Thermomixer (eppendorf, 1,400 rpm) at 37 oC for 90 s. In order to inactivate MNase, EGTA (25 mM) was added immediately after the reaction and soluble-digested chromatin was collected by 13,000 rpm centrifuge for 5 min. The supernatant was diluted with 9 ml of NET-2 buffer (50 mM Tris-Hcl, pH 7.4, 150 mM NaCl, 0.05% NP40) and Pol II antibody-conjugated beads were added. 40 g of Pol II antibody was used for each mNET-seq experiment. Immunoprecipitation was performed at 4 oC for 1 hr. The beads were washed with 1ml of NET-2 buffer six times and with 500 l of 1xPNKT (1xPNK buffer and 0.1% Triton X-100) buffer once in the cold room. The washed beads were incubated in 100 l of PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK 3' phosphatase minus (NEB) on Thermomixer (1,400 rpm) at 37 oC for 6 min. After, the reaction beads were washed with 1ml of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was resolved on 8% denaturing acrylamide 7M urea gels for size purification. 35–100 nt fragments were eluted from the gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) and RNA was precipitated in 75% Ethanol. RNA libraries were prepared according to the manual of NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs).
GEO
GSM4174014
GEO Accession
GSM4174014