SAMD00245767
External Id
Department of Preventive Medicine, Graduate School of Medicine, The University of Tokyo
INSDC center name
2020-09-07T00:00:00Z
INSDC first public
2020-09-10T03:16:25Z
INSDC last update
DRS151951
INSDC secondary accession
live
INSDC status
Generic
NCBI submission model
Generic.1.0
NCBI submission package
DRS151951
SRA accession
polyclonal rabbit IgG (ab37415, Abcam)
antibody
1
biological replicate
RIKEN BioResource Research Center (BRC)
biomaterial provider
MKN1
http://www.ebi.ac.uk/efo/EFO_0002830
cell line
MKN1 human GC cells grown to confluency in eight 15-cm dishes were used for RNA immunoprecipitation (RIP). The cellular protein-RNA complexes were cross-linked by the drop-wise addition of 4% PFA to the culture medium to a final concentration of 0.75% and incubated for 10 mins. The cross-link reaction was stopped by the addition of glycine (G8898, Sigma-Aldrich) to a final concentration of 125 mM and the cells were further incubated for 5 min. The cells were rinsed twice with 10 mL ice-cold PBS and then thoroughly scraped with 5 mL of ice-cold PBS using a cell scraper (MS-93300, Sumitomo Bakelite, Japan). The fixed cells were lysed using a buffer containing 50 mM Tris (Sigma-Aldrich), 150 mM NaCl (FUJIFILM Wako Pure Chemical Corporation), and 0.1% Triton-X100 (Sigma-Aldrich) with proteinase inhibitors (#11873580001, Sigma-Aldrich) and RNase inhibitor (#2313A, Takara Bio, Japan). Debris was excluded by centrifuging at 14,000 rpm for 5 mins at 4degree. The following procedures were carried out with buffer containing RNase inhibitor. To pre-clean non-specific reactions, the initial cell lysate was incubated with 8.0 ug control polyclonal rabbit IgG (ab37415, Abcam) for 1 hr at 4degree; non-specific IgG-protein complexes were removed using protein G beads (#1614023, Bio-Rad Laboratories). The cell lysate was separated into two tubes, each of which was incubated with 1.0 ug control polyclonal rabbit IgG (ab37415, Abcam) overnight at 4degree. The complex was immunoprecipitated by protein G beads (#1614023, Bio-Rad Laboratories) and the protein-RNA crosslinks were reversed by adding 250 mM NaCl (FUJIFILM Wako Pure Chemical Corporation) and 300 ug/mL proteinase K (Sigma-Aldrich) and incubated at 65degree for 1 h with shaking. The precipitated RNA was purified using an RNeasy mini kit (Qiagen). Purified RNA was qualified and quantified using an Agilent Bioanalyzer (Agilent) and rRNA was eliminated using an NEBNext? rRNA Depletion kit (New England Biolabs, MA, USA); a SMART-Seq? Stranded Kit (#634442, Takara Bio) was then used to construct an RNA-seq library according to the manufacturer's protocol.
description
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
MKN1_Ctrl
sample name
2
technical replicate
MKN1_Ctrl
title