title: GSM1474231: mNET_CMA602_siLuc; Homo sapiens; OTHER
library_construction_protocol: Approximately 1.6x108 cells were used to generate nuclear and chromatin fractions. Isolated chromatin was washed in 1 ml of 1x Micrococcal Nuclease (MNase) buffer (NEB) and then incubated with MNase (40 u/mL) on Thermomixer (eppendorf, 1,400 rpm) at 37oC for 90 sec. MNase was inactivated by EGTA (25 mM) addition 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 and add Pol II antibody-conjugated beads. 40 mg of Pol II antibody was used for each mNET-seq experiment. Immuno-precipitation was performed at 4oC for one hour. The beads were washed with 1 ml of NET-2 buffer six times and with 500 ml of 1xPNKT (1xPNK buffer and 0.05 % Triton X-100) buffer once in the cold room. The washed beads were incubated in 100 ml 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 37oC for 6 min. After the reaction the beads were washed with 1 ml of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was resolved on 8 % denaturing acrylamide 7 M 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.