|
| Status |
Public on Mar 22, 2022 |
| Title |
sample5_ccs |
| Sample type |
SRA |
| |
|
| Source name |
HEK293-derived GFP(CAG)91
|
| Organism |
Homo sapiens |
| Characteristics |
cell samples: GFP(CAG)91
genotype: GFP(CAG)91
|
| Growth protocol |
The GFP(CAG)x cell lines were cultured in Gibco™ Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX™, 10% foetal bovine serum (FBS), 100 U ml−1 of penicillin/streptomycin, 15 μg ml−1 blasticidine and 150 μg ml−1 hygromycin. The HD LBCs or their DNA used for SMRT sequencing were obtained from the Coriell BioRepository (Table S1 from Taylor et al - https://doi.org/10.1101/2022.03.08.483398). The LBCs were grown in Gibco™ RPMI with GlutaMAX™ supplemented with 15% Gibco™ FBS (Thermo Fisher), and 1% penicillin-streptomycin. Both the LBCs and the GFP(CAG)x cells were grown at 37 °C with 5% CO2 and tested negative for mycoplasma by Eurofins’ ‘Mycoplasmacheck’ service. GFP(CAG)91 is identical to the previously characterised GFP(CAG)101 but contained a contraction in the cultures used here. Similarly, GFP(CAG)51 had a one CAG expansion compared to when it was first derived and GFP(CAG)308 had a repeat tract above 270 that we could not fully sequence with Sanger sequencing at the time. GFP(CAG)15, GFP(CAG)51, and GFP(CAG)308 are derived from GFP(CAG)101.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The HD LBCs and GFP(CAG)x datasets were generated by first isolating DNA using the Macherey-Nagel Nucleospin™ Tissue Mini kit. PCR products were generated from samples using barcoded primers as listed in Table S1 and Thermo™ Phusion II High Fidelity polymerase. To obtain sufficient quantities of PCR product to proceed with library preparation, multiple identical PCRs were pooled and purified using Macherey-Nagel™ Gel and PCR Clean-up kit columns. The library was generated using the SMRTbell Template Prep Kit (1.0-SPv3) according to manufacturer’s instructions. Samples to be sequenced on the same flowcell were combined in equimolar pools. We loaded between 10 and 12 pM. SMRT sequencing was done using a Sequel at Cardiff University School of Medicine. CCSs were generated from the resulting sequences and processed using SMRT Link.
SMRTbell Template Prep Kit (1.0-SPv3)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Sequel |
| |
|
| Data processing |
FASTQ files converted to FASTA
Repeat Size measured using Repeat Detector
Supplementary files format and content: FASTA files
Supplementary files format and content: Text files containing histograms of repeat size
Library strategy: long read sequencing
|
| |
|
| Submission date |
Mar 19, 2022 |
| Last update date |
Mar 23, 2022 |
| Contact name |
Vincent Dion |
| E-mail(s) |
DionV@cardiff.ac.uk
|
| Organization name |
Cardiff University
|
| Department |
Dementia Research Institute
|
| Street address |
Hadyn Ellis Building
|
| City |
Cardiff |
| ZIP/Postal code |
CF24 4HQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL26180 |
| Series (1) |
| GSE199005 |
Repeat Detector: accurate, efficient, and flexible sizing of expanded CAG/CTG repeats from targeted DNA sequencing |
|
| Relations |
| BioSample |
SAMN26809419 |
| SRA |
SRX14498603 |
