2024-07-14
ENA first public
ERC000011
ENA-CHECKLIST
Department of Molecular Biology, Umeå university
INSDC center name
public
INSDC status
ERS20526850
SRA accession
E-MTAB-14220:p10_RNA
Submitter Id
ArrayExpress
broker name
CRL-1459, ATCC
cell_line
fibroblast
cell_type
not collected
collection date
human
common name
Protocols: Briefly, tonsillar cell suspensions were prepared by tissue homogenizing in RPMI-1640 medium and passed through a 70 µm cell strainer. Red blood cells were lysed using BD PharmLyse lysis buffer according to manufacturer’s instructions. All cell suspensions were frozen in fetal bovine serum (FBS) (Gibco) with 10% DMSO and stored in liquid N2. B cells from tonsils of 5 individuals were enriched using negative selection magnetic beads (EasySep Human B Cell Isolation kit, Stemcell technologies, #17954). They were then pooled to avoid batch effects, with an average viability per donor of 92% (S.D. 1.5%). Enriched B cells were washed in ice-cold ATAC-seq resuspension buffer (RSB, 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2), spun down, and resuspended in 100 mL ATAC-seq lysis buffer (RSB plus 0.1% NP-40 and 0.1% Tween-20 (Thermo Fisher). Lysis was allowed to proceed on ice for 5 min, then 900 mL RSB was added before spinning down again and resuspending in 50 mL 1X Nuclei Resuspension Buffer (10x Genomics). To assess nuclei purity and integrity after lysis, nuclei were stained with Trypan Blue (15250061, Thermo Fisher Scientific), and DAPI (D1306, Thermo Fisher Scientific), according to manufacturer recommendation. If necessary, cell concentrations were adjusted to equal ratios (per donor) prior to starting single-cell GEM emulsion droplet generation with the ATAC-seq NextGEM kit (10x Genomics). Briefly, nuclei were incubated in a transposition mix. Transposed nuclei were then loaded into a Chromium Next GEM Chip J. 9.000 nuclei were loaded per lane, with a target recovery of 5.500 (doublet rate 4% - 4.8%). After GEM emulsion generation, reverse transcription, cDNA amplification and multiome sc library generation occurred, according to manufacturer specifications (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression, CG000338 Rev A, 10x Genomics, September 2020) According to (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression, CG000338 Rev A, 10x Genomics, September 2020)
description
infant
developmental stage
normal
disease
not collected
geographic location (country and/or sea)
not applicable
isolate
RNA
modality
Homo sapiens
organism
Homo sapiens
organism
colon
organism part
10
passage
Homo sapiens
scientific_name
p10_RNA
title