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Sample GSM4984126

Query DataSets for GSM4984126

Status

Public on Mar 01, 2021

Title

BR.a8

Sample type

SRA

Source name

FAC-sorted pDCs from Donor 2

Organism

Homo sapiens

Characteristics

donor number: Donor 2
cell type: pDCs
treatment: CMV-infected MRC5 cells
time: 2 hr

Treatment protocol

Cells were stimulated or co-cultured with 2.5 uM CpG, IL-3, 10 MOI CMV, MRC5 cells, or CMV-infected MRC5 cells for 2, 4, 8, 16, or 32 hours.

Growth protocol

PBMCs from donors' blood were isoalted by Ficoll gradient (GE Healthcare). pDCs and pan-DCs wre enriched from PBMCs by negative selection using EasySep Human Plasmacytoid DC Enrichment Kit and EasySep Human pan-DC Pre-Enrichment Kit (STEM CELL Technologies) with ~80-90% purity and cultured in RPMI-1640 complete medium with 2 mM L-gluatamine, 1 mM sodium pyruvate, 1x MEM non-essential amino acids, 100 U/mL penicillin-streptomycin, and 10% fetal bovine serum. MRC5 human lung fibroblasts were cultured under the same conditions. All cells were incubated at 37C with 5% carbon dioxide.

Extracted molecule

polyA RNA

Extraction protocol

pDCs were detached from plates by gentle pipetting and stained for viability with the Zombie Aqua Fixable Vaiability Kit in PBS for 20 mins at 4C. Cells were washed in 4C medium and FAC-sorted to remove MRC5 cells based on forward and side-scatter using an SH800Z sorter (SONY Biotechnology). Total RNA from sorted live pDCs was isolated using an RNeasy Mini Kit (QIAGEN).
Pooled RNA-seq was performed using the low-input RNA-seq protocol reported in Snyder et al, Science Immunology, 2019. All of the steps prior below that come before the barcoded cDNA libraries are pooled were performed using a BioMek 4000 automated liquid handling robot (Beckman). Briefly, we added deoxyribonucleotides (dNTPs) (ThermoFisher) to a final concentration of 2 mM and reverse transcriptase primer to 2 uM followed by primer annealing at 72C for 3 mins. We then added 7.5 uL of reverse transcription mixture to each well comprised of 2M betaine (Affymetrix), 2x ProtoScript Buffer (NEB), 12 mM MgCl2 (ThermoFisher), 10 mM dithiothreitol (ThermoFisher), 5.3 U of Protoscript II reverse transcriptase (NEB), 0.53 U of SUPERaseIN (ThermoFisher), and 2 uM template-switching oligo. The reverse transcription reaction was incubated at 42C for 90 mins followed by 10 cycles of 50C for 2 mins, 42C for 2 mins, and 70C for 10 mins. Next, 2 uL of exonuclease I (ThermoFisher) was added to each well (1.875 U of exonuclease I in water) and the resulting mixture was incubated at 37C for 30 mins, 85C for 15 mins, and 75C for 30s.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw data obtained from the Illumina NextSeq 500 was trimmed and aligned as described in Yuan et al, Genome Medicine, 2018. For each read with a unique, strand specific alignment to exonic sequence, we constructed an address comprised of the sample barcode, unique molecular identifier (UMI) barcode, and gene identifier. The resulting address file only contained the addresses from reads with corresponding single index. We generated a digital gene expression matrix and filter sample barcodes as described previously (Yuan et al. 2018). Briefly, reads with the same sample barcode, UMI and aligned gene were collapsed and sequencing errors in the sample barcode and UMI were corrected to generate a matrix of molecular counts for each cell.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text file that contains the gene expression counts of each sample.

Submission date

Dec 22, 2020

Last update date

Mar 01, 2021

Contact name

Peter A Sims

E-mail(s)

pas2182@columbia.edu

Organization name

Columbia University

Street address

3960 Broadway, Lasker 203AC