2024-09-01
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA113443694
External Id
IMPERIAL COLLEGE LONDON
INSDC center name
2023-05-16T10:48:18Z
INSDC last update
public
INSDC status
ERS15438800
SRA accession
E-MTAB-12944:Sample 2
Submitter Id
ArrayExpress
broker name
HeLa
cell_line
human
common name
Protocols: HeLa cells were maintained in RPMI 1640 or DMEM medium, respectively, supplemented with 10% foetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin at 37°C in 5% CO2 in a humidified incubator. AGO2-eiCLIP was performed as previously described (Paterson et al. 2022). In brief, this involved following a previously described non-isotopic iCLIP workflow (Sibley 2018) which had additional modifications to enhance speed and efficiency. This included ligation of a new Cy5.5 labelled adapter (/5Phos/A[XXXXXX]NNNAGATCGGAAGAGCACACG/3Cy55Sp/) to bound RNA with high concentration T4 RNA ligase (New England Biolabs), use of RecJf exonuclease (New England Biolabs) to remove un-ligated adapter prior to SDS-PAGE analysis, reverse transcription with a biotinylated primer (/5BiotinTEG/CGTGTGCTCTTCCGA), exonuclease III (New England Biolabs) mediated removal of unextended RT-primer, cDNA capture with MyOne C1 streptavidin beads (ThermoFisher Scientific), 3’ adapter (/5Phos/ANNNNNNNAGATCGGAAGAGCGTCGTG/3ddC/) ligation instead of intramolecular ligation, and cDNA elution with nuclease and cation free water at high temperature. For a pellet of cells obtained from a 80% confluent 150mm dish, we used 100 µl Dynabeads Protein G (Thermo Fisher Scientific) conjugated to 1.5 µg anti-AGO2 antibody (MAB253, Sigma-Aldrich/ Merck). Samples of two biological replicates were sequenced with paired-end reads using NextSeq500.
description
adult
developmental stage
Homo sapiens
organism
Homo sapiens
organism
uterine cervix
organism part
bio rep2
replicate
Homo sapiens
scientific_name
Sample 2
title