2024-07-04
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA115084289
External Id
Institut Cochin, INSERM U1016, CNRS UMR8104, U.Paris-Cité
INSDC center name
2024-01-05T17:23:13Z
INSDC last update
public
INSDC status
ERS17700262
SRA accession
E-MTAB-13689:INP_Myo_shCTL
Submitter Id
ArrayExpress
broker name
immortal cell line cell
cell_line
skeletal muscle myoblast
cell_type
not collected
collection date
human
common name
Protocols: All samples correspond to immortallized human myoblast cell line. The cells expressing shZNF55 and non targeting shCtl were compared. Cells were washed in PBS and were mixed with 500 µl of ice-cold lysis buffer (Quiagen), incubated for 5 min and centrifuged et 10 000 rpm for 5 min. The supernatant was collected and then diluted in 300 µl of LDS extraction buffer (glycerol 10%, Tris-Base (0.14 M), Tris-HCl (0.14M), LDS (2%), EDTA 500nM). The samples were shortly sonicated to reduce the viscosity and then centrifuged. The obtained supernatant was mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1 [vol/vol]). The aqueous phase was retained by centrifugation, and then the dissolved DNA was precipitated with 0.1 volume of NaCl 2.5 M and 2.5 times greater volume of ice-cold ethanol. The samples were then centrifuged at 15000 × g for 15 min at 4°C. Finally, the pellet of DNA was washed with pre-cooled 70% ethanol and resuspended in 50 μL Milli-Q water. Concentration of the extracted DNA was measured using the Qubit instrument and the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The genomic DNA was sonicated (approximately 300-700 bp in size) and MIRA pull-down was performed using the Methyl Collector Ultra Kit (Active Motif) per manufacturer’s instructions starting with 1 µg of DNA and using high salt binding conditions. Extracted genomic DNA (gDNA) was sonicated in a Covaris S220 (LGC Genomics GmbH) to a mean fragment size of 180 bp. Fragment length distribution was assessed by microelectrophoresis using the Bioanalyseur 2100 (Agilent). Enriched DNA and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific) using the Bravo Platform (Agilent) and amplified by PCR for 10 cycles. The final libraries were purified with AMPure XP beads (Beckman Coulter).
description
adult
developmental stage
non target shRNA
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
skeletal muscle organ
organism part
Homo sapiens
scientific_name
INP_Myo_shCTL
title