|
| Status |
Public on Jan 25, 2023 |
| Title |
K9minus2_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
RNA-seq (Poly A minus)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood cells
cell type: Differentiated erythroid cells
genotype: HbE/beta thalassaemia
treatment: Control
|
| Growth protocol |
CD34+ cells were resuspended in Phase I medium for a three-phase differentiation adapted from that used for the BEL-A cell line (Trakarnsanga, Nat Comms 2017). Briefly, for differentiation 3x10^5 cells were resuspended on day 0 in Phase I media at 2x10^5 cells ml-1. Cell counts were performed on days 3 and 5 with additional Phase I media added to return the concentration to 105 cells ml-1. On day 7, cells were counted and pelleted (300 rcf, 5 min, RT) and resuspended in Phase II media at 2x10^5 cells ml-1. Cells were counted on day 9 and diluted to 2x10^5 cells ml-1 Phase II media. HUDEP-2 cells were cultured in expansion phase as previously described Canver, M. C. et al.,2015.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermofisher) following the manufacturer's protocol with no modifications
For sequencing of base edited sites, modified 2-PCR version of the NEBnext Ultra (NEB) library preparation protocol was followed. In the first PCR primer pairs with NEB adaptor sequences 5’ to the HBB exon 1-specific primer sequence were used for amplification using the Herculase II PCR kit (Agilent) in a 50μl reaction, ensuring the products had the adaptor sequences added by the end of the PCR. 5μl of this was used directly without clean-up for the indexing PCR. This added either single or dual end indices taken from a NEBNext Multiplex Oligos for Illumina kit (NEB).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
RNA-seq
RNAseq_splicing.txt
|
| Data processing |
Base editing data were analysed with CRISPRESSO
RNA-seq data was aligned using STAR (2.7.3a) to hg19. Aberrant splicing of Poly-A negative RNA was detected using PySam (https://github.com/pysam-developers/pysam) find_introns method, considering position 5,248,159 of chromosome 11 (hg19) as the canonical splice site for exon 1 of the HBB gene. An RNA variant calling pipeline was established using GATK best practices. In keeping with the Broad Institutes recommendation for this tool, these data were realigned to hg38 using STAR two-pass alignment, followed by PCR duplicate removal and base score recalibration. GATK Haplotype Caller (4.0.11.0) was then used to call variants.
Assembly: Hg19
Supplementary files format and content: Base_Editing_Frequencies.txt
Supplementary files format and content: RNAseq_splicing.txt
|
| |
|
| Submission date |
Jan 24, 2023 |
| Last update date |
Jan 25, 2023 |
| Contact name |
James Oliver Joshua Davies |
| Organization name |
Weatherall Institute of Molecular Medicine, Oxford University
|
| Department |
Molecular Haematology Unit
|
| Lab |
Davies
|
| Street address |
Weatherall Institute of Molecular Medicine, John Radcliffe Hospital
|
| City |
Oxford |
| State/province |
Oxon |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE206098 |
Direct correction of haemoglobin E / beta-thalassaemia using base editors |
|
| Relations |
| BioSample |
SAMN32901638 |
| SRA |
SRX19162954 |
