|
| Status |
Public on Mar 01, 2017 |
| Title |
Sample ERCC2 capture replicate 2 with 1.0 ng cDNA input |
| Sample type |
SRA |
| |
|
| Source name |
ERCC RNA mixed with PBMC RNA
|
| Organisms |
Homo sapiens; synthetic construct |
| Characteristics |
cell type: ERCC/PBMC
amount cdna (ng) input used for smmip capture: 1
number of pcr cycles: 20
cdna sample id: ERCC2-cDNA
|
| 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 |
The synthetic ERCC controls (Thermo Fisher Scientific, Waltham, MA, USA) were diluted 1:100, and 46 ul was used for cDNA synthesis after mixing with human PBMC total RNA using Superscript III Reverse Transcriptase (Thermo Fisher Scientific). Samples were purified using Qiaquick (Qiagen, Venlo, The Netherlands) columns, and cDNA was quantified using the Qubit ssDNA assay (Thermo Fisher Scientific). Of each sample 1 and 2 ng cDNA was used for MIP capture, and all capture experiments were performed in duplo.
processed data file:
all_samples_molecule_counts.txt
cdna_mips_ercc.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
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| |
|
| 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
|
| Street address |
Geert Grooteplein 10
|
| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL23058 |
| Series (1) |
| GSE94800 |
Quantification of differential gene expression by multiplexed targeted resequencing of cDNA |
|
| Relations |
| BioSample |
SAMN06322238 |
| SRA |
SRX2553255 |
