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Status |
Public on Mar 22, 2022 |
Title |
sample1_sub11_unfilteredCCS |
Sample type |
SRA |
|
|
Source name |
Lymphoblastoid cell line
|
Organism |
Homo sapiens |
Characteristics |
cell samples: NA20251 genotype: HD
|
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)
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|
|
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
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|
|
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 |
SAMN26809433 |
SRA |
SRX14498589 |