MBE-03587
Protocols: Following treatment, cells were washed once with PBS directly in 384-well plates and lysed in-well for downstream reverse transcription. No physical dissection was performed as samples consisted of cultured cell lines. Human iPSC lines (MBE-03587 and MBE-03590; PMID: 37657710) were maintained onto Vitronectin XF™-coated plates (Stem Cell Technologies) in StemFlex™ (Thermo Fisher Scientific), with media changes every second day and weekly passaging using ReLeSR™ (Stem Cell Technologies). iPSCs were differentiated in E6 medium (Stem Cell Technologies) supplemented with N2 supplement (Life Technologies) and penicillin–streptomycin (Life Technologies) for 30 days then in RPEM (αMEM, 5% FBS, non-essential amino acids, N1 supplement, penicillin-streptomycin-glutamate, taurine-hydrocortisone- triiodothyronine) for 30 days, passaged into their final format by automation.   Cells were treated with Mitoquinone, Calmidazolium, Corynoline, Latrepirdine, and Visomitin at concentrations of 0.1 µM or 1 µM. Staurosporine (1 µM and 10 µM) was included as a cytotoxic control. DMSO vehicle controls and untreated wells were included.  RNA was captured directly from cell lysates using the MAC-seq protocol. Reverse transcription was performed at 42°C for 2 hours. Resulting cDNA was pooled and purified using Zymo DNA Clean & Concentrator columns followed by AMPure XP bead cleanup according to manufacturer protocols. Amplified cDNA was generated by pre-amplification PCR and purified using AMPure XP beads. cDNA quality and fragment size distribution were assessed using Agilent D5000 ScreenTape. Libraries were prepared by shearing 100–500 ng cDNA to 300–350 bp fragments using a Covaris ME220 system. Fragmented cDNA underwent end repair, adaptor ligation, size selection using AMPure XP beads, and index PCR amplification. Final libraries were purified using AMPure XP beads and assessed using Agilent D1000 ScreenTape. Libraries were quantified using qPCR with the KAPA Library Quantification Kit.
ENA first public
2026-07-31
INSDC center name
The University of Melbourne \
INSDC status
public
Submitter Id
E-MTAB-16981:MBE-03587
broker name
ArrayExpress
cell line
MBE-03587
cell type
retinal pigment epithelial cell
collection date
not collected
common name
human
disease
age-related macular degeneration
genotype
wild type genotype
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
retinal pigment epithelium
sample name
E-MTAB-16981:MBE-03587
scientific_name
Homo sapiens
strain
MBE-03587