2024-05-31
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA113565704
External Id
UNIVERSITY OF MANCHESTER
INSDC center name
2023-05-31T11:23:59Z
INSDC last update
public
INSDC status
ERS15561852
SRA accession
E-MTAB-13014:CellPlex iBMDMs (CML)
Submitter Id
ArrayExpress
broker name
BMDMs
cell_line
macrophage
cell_type
house mouse
common name
Protocols: Fluorescence activated cell sorting (FACS) was used to sort single iBMDMs into prewarmed DMEM+FBS in 96 well plates and incubated to generate clonal populations. To reduce clumping during growth from single cells, cells were dispersed by gently reverse pipetting on days 5 and 8. When cells were 60-80% confluent (day 12-14) clonal populations were transferred to 6 well plates for subsequent experiments. Clonal or mixed populations in 6 well plates at approximately 50% confluence were stimulated with Lipid A at a final concentration of 500ng/ml for 3h. Cells were harvested by scraping and put on ice prior to tagging using the 10x genomics 3' CellPlex kit according to the manufacturer's instructions for >80% viable cells (protocol 1 document number CG000391). Gene expression and Cell Multiplexing libraries were prepared from Cell Multiplexing Oligo labelled cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer’s protocol (CG000388 Rev B). Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a Chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16-nucleotide 10x Barcode, a 12-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents along with primers containing an Illumina Nextera Read 1, a 16-nucleotide 10x Barcode, a 12-nucleotide UMI and a capture sequence. Incubation of the GEMs yielded barcoded cDNA from poly-adenylated mRNA and barcoded DNA from the Cell Multiplexing Oligo Feature barcode. Following incubation, GEMs were broken and pooled fractions recovered. The cell barcoded cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library constructions. The amplified cDNA molecules for 3’ Gene Expression were separated from those for Cell Multiplexing library construction by size selection.For the 3’ Gene Expression library enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5, P7, i7 & i5 sample indexes, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.For the Cell Multiplexing library P5, P7, i7 & i5 sample indexes and Nextera Read 2 were added via PCR to amplified DNA from Cell Multiplexing Oligo Feature Barcodes to yield final Illumina-compatible sequencing libraries
description
wild type genotype
genotype
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
bone marrow
organism part
multiplexing capture
protocol
Mus musculus
scientific_name
female
sex
C57BL/6
strain
CellPlex iBMDMs (CML)
title