2024-12-09
ENA first public
ERC000011
ENA-CHECKLIST
University of Cambridge
INSDC center name
public
INSDC status
ERS20522256
SRA accession
E-MTAB-14219:RPE-1
Submitter Id
ArrayExpress
broker name
RPE-1
cell_line
retinal pigment epithelial
cell_type
not collected
collection date
human
common name
Protocols: Cells were seeded on 10cm plates for 48 hours. To harvest, cells were washed once in PBS, incubated with cytoplasmic lysis buffer (50 mM HEPES pH7.9, 10 mM KCl2, 1.5 mM MgCl2, 0.34 M sucrose, 0.5% triton, 10% glycerol, 1 mM DTT and 0.1mg/mL RNase A) for 10 minutes at room temperature, washed once in cytoplasmic lysis buffer and finally scraped in genomic extraction buffer (50mM Tris pH 8.0, 5mM EDTA, 1% SDS, 0.5mg/mL proteinase K (Thermo, 25530049).  Lysates were transferred to 2mL DNA LoBind microcentrifuge tubes (Fisher Scientific, 0030108426) and incubated at 60 °C with 500rpm agitation for 1 hour. Sodium acetate pH 5.2 was added to a final concentration of 300mM and 2.5 volumes of 100% ethanol was added. Samples were incubated on ice for 1 hour to precipitate the genomic DNA and pelleted at 21,000g for 20 minutes at 4 °C. Genomic DNA pellets were washed once in 75% ethanol and re-pelleted for 10 minutes before being allowed to air-dry and be resuspended in nuclease free water.  DNA samples were diluted to 100ng/µL in 300µL in 1.5mL Bioruptor Plus TPX Microtubes (Diagenode, C30010010-300) and sonicated in a Bioruptor Plus sonicator for 20 cycles of 30 seconds on/30 seconds off with active chilling at 4 °C, shearing the DNA to an average length of ~600-800bp. 500ng of sheared genomic DNA was used for library preparation using the IDT xGen DNA library preparation kit (IDT, at 10009861) coupled to the xGen Exome Hyb Panel v2 for exome enrichment (IDT, 10005151) all according to the manufacturer instructions.
description
N/A
developmental stage
N/A
disease
not collected
geographic location (country and/or sea)
RPE-1
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
Homo sapiens
scientific_name
RPE-1
title