barcode_I
Homo sapiens
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.
ENA first public
2025-10-30
INSDC center name
UniTo
INSDC status
public
Submitter Id
E-MTAB-15332:barcode_I
broker name
ArrayExpress
cell line
WTC11
cell type
iPS2-seq-hiPSC
collection date
not collected
common name
human
developmental stage
NA
disease
normal
genotype
Barcodes KD screening in pool (multiple genotypes)
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
hiPSC
sample name
E-MTAB-15332:barcode_I
scientific_name
Homo sapiens