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Sample GSM2483356 Query DataSets for GSM2483356
Status Public on Mar 01, 2017
Title Sample K562HEK-3 capture replicate 2 with 10.0 ng cDNA input
Sample type SRA
Source name K562 and HEK293 cell line
Organism Homo sapiens
Characteristics cell type: K562/HEK293
amount cdna (ng) input used for smmip capture: 10
number of pcr cycles: 22
cdna sample id: 3-2-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 Culturing of HEK cell line: HEK293T cells (ECACC 12022001) were cultured in 100mm tissue-culture treated culture dishes (Corning, New York, USA) in DMEM medium containing 10% (vol/vol) Fetal Bovine Serum 1% penicillin/streptomycin and 1% sodium pyruvate (all Sigma-Aldrich, St. Louis, USA). At passage 12, cells were detached using 0.25% Trypsin (BD Biosciences, San Jose, USA) after which ~3 million cells were used for RNA isolation. Culturing of K562 cell line: K562 cells (ECACC 89121407) were cultured in 75 cm2 cell culture flasks (Corning, New York, USA) in RPMI 1640 medium containing 15% Fetal Bovine Serum, 2% HEPES and 1% penicillin/streptomycin (all Sigma-Aldrich, St. Louis, USA). ~6 million cells were used for RNA isolation. Allelic ratio dilution curve: cDNA of K562 and HEK293T cells was combined in a serial dilution. cDNA of K562 and HEK293T cells was diluted to a final concentration of 1 ng/ul. The serial dilution started with 75% K562 cDNA and 25% HEK293T cDNA. For the following steps of the serial dilution, 75% of the cDNA from the previous step was combined with 25% HEK293T cDNA. This resulted in a series of 8 cDNA samples of which the concentration K562 cDNA exponentially decreased and the HEK293T cDNA increased accordingly. One sample with only K562 cDNA and one sample with only HEK293T cDNA were also included in the experiment. Subsequently, samples were divided into two 10 ul duplicates containing 10 ng cDNA each. Capture was performed using the allelic-ratio smMIPS.
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 SAMN06322263
SRA SRX2553313

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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