2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
UniTo
INSDC center name
public
INSDC status
ERS25146962
SRA accession
E-MTAB-15332:GEX_I
Submitter Id
ArrayExpress
broker name
WTC11
cell line
iPS2-seq-hiPSC
cell type
not collected
collection date
human
common name
Protocols: Cells were detached using Versene and incubated with DNase I (200 U/mL) to reduce clumping, then washed and filtered through a 40 µm strainer (Flowmi). Cell viability and count were assessed using trypan blue staining (Sigma) on a hemocytometer. Sample preparation for single-cell multiome sequencing was performed following the 10X Genomics demonstrated protocol for nuclei isolation (CG000365, Rev D) and the Multiome User Guide (CG000338, Rev F). HiPSC cells transfection pools were kept separately, and nuclei were pooled only prior to loading on the 10X Chromium. After cell lysis using freshly prepared buffer containing 0.1% Tween-20, IGEPAL CA-630, 0.01% digitonin, 1% BSA, and 1U/ml RNAse inhibitor, nuclei were isolated, washed twice with chilled wash buffer, and counted using both trypan blue and 1:1000 YOYO-1 staining (Invitrogen). Final nuclei counts were pooled 1:1 and diluted to 7,000 nuclei/μL before loading into the microfluidic chip. Nuclei were loaded after diluting in 10X Nuclei buffer according to the user guide. Chromatin tagmentation and reverse transcription were performed according to the 10X protocol. Libraries were indexed and amplified: 7 cycles for ATAC index PCR and 6 cycles for cDNA amplification. D5000 Tapestation and dsDNA hi-sensitivity Qubit analyses revealed appropriate fragment sizes and concentrations. The iPS2-seq library was obtained from the cDNA of the 10X gene expression library, as outlined in the protocol section. Adaptations of the current protocol were necessary to obtain clean iPS2-seq libraries from the multiome cDNA library. Reduced cycle amplification to 11 cycles and increased input material to 200ng was key to this optimization, resulting in libraries with cleaner profiles, confirmed by D5000 Tapestation analysis and improved size distributions following 1.8x–0.7x SPRIselect purification. Libraries were further amplified and indexed using TT indices, and purified again using a double-sided size selection.
description
pluripotency
developmental stage
normal
disease
KD screening in pool (multiple genotypes)
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
hiPSC
organism part
Homo sapiens
scientific_name
GEX_I
title