ERC000011
ENA-CHECKLIST
2023-12-11T00:33:11Z
ENA-FIRST-PUBLIC
2023-12-11T00:33:11Z
ENA-LAST-UPDATE
SAMEA113542155
External Id
THE UNIVERSITY OF TOKYO
INSDC center name
2023-12-11T00:33:11Z
INSDC first public
2023-12-11T00:33:11Z
INSDC last update
public
INSDC status
ERS15538572
SRA accession
E-MTAB-12988:Sample 1: DNA barcodes of micro-beads in ID-coded droplets (with single cells)
Submitter Id
ArrayExpress
broker name
mixed K562 and HEK293
cell_line
Protocols: HEK293 cells were purchased from JCRB CellBank (JCRB9068); K562 cells were purchased from JCRB CellBank (JCRB0019). Murine NIH/3T3 cells (spiked only in Sample 1) were purchased from JCRB CellBank (JCRB0615). Micro-particles functionalized both by DNA barcodes and fluorescent molecules were synthesized and used for ID-coding. HEK293 cells were purchased from JCRB CellBank (JCRB9068); K562 cells were purchased from JCRB CellBank (JCRB0019). Murine NIH/3T3 cells (spiked only in sample 1) were purchased from JCRB CellBank (JCRB0615). K562 cells were grown in RPMI1640 medium (Sigma-Aldrich) supplemented with 10%FBS (Sigma-Aldrich) and 1x Antibiotic Antimycotic (Thermo Fisher Scientific). HEK293 cells were grown in Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 10% Horse serum (Thermo Fisher Scientific) and 1x Antibiotic Antimycotic to reach 60-80% confluence. NIH/3T3 cells (used to estimate the rate of doublet errors during NGS library preparation, Fig. S5a) were grown in Dulbecco’s Modified Eagles’ Medium low glucose (Sigma-Aldrich) supplemented with 10% Newborn Calf serum (Thermo Fisher Scientific) and 1x Antibiotic Antimycotic to reach 60-80% confluence. Cell pellets were resuspended in a buffer containing 0.75% (w/v) agarose (Sigma Aldrich, ultra-low gelling temperature). The suspension was passed through pluriStrainer (20 μm), mixed with pellets of DNA-barcoded microbeads. Monodisperse droplets were generated by flowing the beads suspension and HFE7500 oil supplemented with 2% 008-fluorosurfactant (RAN Biotechnologies) into a droplet generation chip. After droplet generation, water-in-oil hydrogel droplets were cooled at 4 °C at 500 rpm shaking. To the droplets, medium-based buffer solution and perfluoro octanol (final volume: 20% in oil, v/v) were slowly added, and hydrogel droplets were extracted into the aqueous layer, which was further washed with 20% perfluorooctanol in HFE7200, and hexane supplemented with Span 80. The resulting hydrogel droplets were firstly subjected to microscopic imaging and then “single-droplet” sequencing, where the whole nucleic acid components in a hydrogel droplet were processed as single-cell components in the droplet-based single-cell RNA sequencing library preparation. Cell lysis and RNA extraction using polyT-functionalized DNA-barcoded beads for droplet-based single cell RNA sequencing (drop-seq) Reverse transcription and cDNA preparation using polyT-functionalized DNA-barcoded beads for droplet-based single cell RNA sequencing (drop-seq)
description
mixed adult and embryo
developmental stage
normal
disease
DNA barcoding
library construction
synthetic construct
organism
synthetic construct
organism
mixed bone marrow and kidney
organism part
synthetic construct
scientific_name
Sample 1: DNA barcodes of micro-beads in ID-coded droplets (with single cells)
title