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Sample GSM2483380 Query DataSets for GSM2483380
Status Public on Mar 01, 2017
Title Sample PBMC-RPMI capture replicate 2 with 50.0 ng cDNA input
Sample type SRA
Source name Peripheral blood mononuclear cells
Organism Homo sapiens
Characteristics cell type: PBMC
amount cdna (ng) input used for smmip capture: 50
number of pcr cycles: 21
cdna sample id: PBMC-RPMI-cDNA-1
Treatment protocol Except for the PBMC stimulation experiment, cells were not stimulated. In the PBMC experiment, cells were stimulated with heat-killed Candida Albicans.
Growth protocol Primary cells and cell lines were grown according to the description provided for each sample.
Extracted molecule total RNA
Extraction protocol The isolated total RNA was quantified using the Qubit RNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis of 2-5 µg of RNA was performed with iScript (BIO-RAD, Hercules, CA, USA) reverse trancriptase. After cDNA synthesis, the cDNA was purified using Qiaquick (Qiagen, Venlo, The Netherlands) purification columns, and cDNA quantity was measured using the Qubit ssDNA assay kit (Thermo Fisher Scientific).
Single-molecule inversion probes applied to cDNA. Main steps are 1) MIP phosphorylation 2) MIP capture 3) Exonuclease treatment; 4) PCR amplification
Targeted RNA-Seq using cDNA-smMIPs
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description Isolation and stimulation of peripheral blood mononuclear cells (PBMC) was performed as previously described (van de Veerdonk et al). In short, venous blood was collected into EDTA tubes and primary blood mononuclear cells (PBMCs) were isolated by density centrifugation of blood diluted 1:1 in PBS over Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). Cells were washed three times in PBS and resuspended in RPMI 1640 (Dutch modified) supplemented with 50 mg/L gentamicin, 2 mM l-glutamine, and 1 mM pyruvate. Cells were counted in a Coulter Counter Z (Beckman Coulter, Mijdrecht, the Netherlands) and adjusted to 5x10^6 cells/mL. Mononuclear cells (5x10^7) in a 2 mL volume were added to round-bottom 6-well plates (Greiner, Alphen a/d Rijn, the Netherlands) and incubated with either culture medium (negative control) or heat killed (HK) Candida (1x106/mL) for 24 hours before isolation of RNA using RNeasy mini kit (Qiagen, Venlo, The Netherlands).
processed data file:
Data processing Match each read pair to a designed smMIP probe (allowing two mismatches);
Remove likely extension-ligation dimers;
For all reads assigned to a given smMIP probe, identify the molecule counts from the unique molecular identifiers;
Determine the read-dependent threshold for UMIs due to sequencing errors;
Estimate normalized expression levels from error-corrected molecule counts integrating replicates using a dedicated Bayesian model.
Genome_build: Reads for the HEK/K562 allelic ratio experiment were mapped to hg19. For all other experiments, no read mapping was performed.
Tab-delimited file with molecule counts for each smMIP and sample, corrected and uncorrected for sequencing errors in the molecular identifiers.
all_samples_molecule_counts.txt: txt file with molecule counts for all experiments
cdna_mips_ercc.txt: txt file with probe design for ERCC experiments
cdna_mips_geuvadis.txt: txt file with probe design for PBMC and EBV experiments
cdna_mips_ase-K562-HEK293.txt: txt file with probe design for allelic ratio experiments
Submission date Feb 10, 2017
Last update date May 15, 2019
Contact name Kees Albers
Organization name Radboud University Medical Center
Department Human Genetics
Street address Geert Grooteplein 10
City Nijmegen
ZIP/Postal code 6525GA
Country Netherlands
Platform ID GPL18573
Series (1)
GSE94800 Quantification of differential gene expression by multiplexed targeted resequencing of cDNA
BioSample SAMN06322185
SRA SRX2553337

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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