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Sample GSM2483351

Query DataSets for GSM2483351

Status

Public on Mar 01, 2017

Title

Sample K562HEK-1 capture replicate 1 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: 1-1-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:
all_samples_molecule_counts.txt
cdna_mips_ase-K562-HEK293.txt

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.
Supplementary_files_format_and_content:
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

E-mail(s)

kees.albers@radboudumc.nl

Organization name

Radboud University Medical Center

Department

Human Genetics