title: GSM2409889: siNEAT1_V2-1; Homo sapiens; ncRNA-Seq
library_construction_protocol: Total RNA was extracted by TRIZOL reagent.  For CLIP-seq, HeLa cells were UV irradiated at 400 mj and collected by scraping the cells from 15 cm plates. Cell lysis was treated by RQI DNase to digest genomic DNA and the supernatant was collected for subsequent immunoprecipitation. For each immunoprecipitation, 2µg NONO or PSF antibody was incubated with 200µl Dynabeads (Invitrogen) for 2hrs at 4℃. Then, cell lysis was added to the washed beads and rotated with the beads for another 2-4hrs at 4℃. 3' linker was ligated to RNA on the Immunoprecipitated protein-RNA complex. After western blotting onto nitrocellulose, recovery of protein-RNA complex, and removal of proteins by proteinse K digestion, a 5'linker was ligated to RNA. Isolated total RNA (5-8 μg) was mixed with 10 μM pre-adenylated 3’ linker in 1x RNA ligase buffer containing 50% (w/v) PEG 8000, 1 μl RNase inhibitor, and 1 μl T4 RNA ligase 2 (NEB), and incubated at room temperature for 1 hrs, then at 16℃ for 3 hrs.  After 3’ linker ligation, the reaction was size fractionated on 15% polyacrylamide gel and RNAs between 42 to 54 nt were recovered by incubating the corresponding gel slice at 4oC in 600 μl of 0.3 M NaCl with constant agitation.  5’ linker ligation was next performed in 1x RNA ligase buffer containing 50% (w/v) PEG 8000, 1 mM ATP, 10 μM 5’ linker, 1 μl RNase inhibitor and 1 μl T4 RNA ligase 1 (NEB) at 37℃ for 4 hrs.  Linker-ligated RNAs were reverse transcribed with Superscript III (Life Technology) and PCR-amplified with Phusion polymerase (ThermoFisher).  The product was fractionated on 4% agarose gel and recovered DNA was quantified for deep sequencing.  And for CLIP-seq, immunoprecipitated RNA was extracted using Trizol, and after DNase I (Promega) treatment, RNA was reverse transcribed into cDNA using superscript III with random primer. The resulting cDNA was quantified for deep sequencing. small RNA-seq and CLIP-seq
attributes.tag: GEO Accession
attributes.value: GSM2409889