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| Status |
Public on May 29, 2024 |
| Title |
PB CD34+ HSPCs, SF3B1 mutant, , SMARTseq |
| Sample type |
SRA |
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| Source name |
Hematopoietic
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Hematopoietic
cell type: CD34+ HSPCs from mobilized peripheral blood
genotype: SF3B1 K700E/+
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| Treatment protocol |
HSPCs were CRISPR-edited using the Lonza 4D Nucleofection system and the Human Primary Cell Nucleofection Kit (Lonza). RNP complexes were generated by combining Cas9 protein and sgRNA (Synthego) at 1:2.5 molar ratio in P3 Primary Cell Nucleofector Solution with Supplement 1 (Lonza). CD34+ HSPCs were electroporated using Lonza 4D (DZ-100 program) and returned to HSPC media containing AAV particles comprising < 20% culture volume, as recommended. HSPCs were washed 16-24 hours later and plated in HSPC media for 48 hours.
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| Growth protocol |
HSPCs were cultured in StemSpan SFEM II (StemCell Technologies), 100 U/mL penicillin/streptomycin (Fisher), 100 ng/ml SCF, 100 ng/ml FLT3, 100 ng/ml TPO, 100 ng/ml IL-6 (all Peprotech), 35 nM UM171 (Apexbio Technology) and 0.75 μM SR1 (Cellagen Technology). Cell density was maintained between 4 x 10^5 and 1 x10^6 per ml.
SF3B1-mutant (K700E) and wild-type control K562 cells (25) were grown in Iscove’s modified Dulbecco’s media (IMDM) with 10% FBS and 100 U/mL penicillin/streptomycin (Fisher), and cultures were maintained at <1.5 x 10^6 cells/ml.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were resuspended in Trizol (ThermoFisher, cat. #15596026), and RNA extracted per manufacturer protocol.
RNA sequencing was performed at Omega Bioservices with TruSeq Stranded mRNA kit with polyA selection. PB BFP+CD34+ gene edited HSPCs were additionally sequenced with SMARTSeq v4 Ultra Low Input RNA Kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
SMARTSeq v4 Ultra Low Input RNA Kit
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| Data processing |
For gene expression analysis, reads were pseudoaligned using kallisto.
Differential gene expression was determined using the DESeq2 package.
For splicing analysis, RSEM v1.2.4 was used to map all RNA-seq reads to this transcriptome annotation.
Remaining unaligned reads were then mapped a database of all possible junctions between annotated 5' and 3' splice sites of the transcriptome annotation, as well as to the genome sequence, with TopHat v2.0.8b, and the resulting aligned reads were merged with the RSEM output
The expression levels of isoforms annotated in MISO v2.0’s annotation were estimated with MISO v2.0.
To identify events that were differentially spliced, we defined the metric deltaPSI (ΔPSI) as the isoform ratio (absolute percentage of mRNA) in SF3B1-mutant samples - isoform ratio in SF3B1-WT cells, and computed a significance statistic using Wagenmakers’s Bayesian framework for single-sample comparisons or the Mann-Whitney U test for group comparisons. An event was classified as differentially spliced if it exhibited an absolute change of ΔPSI ≥ 10% and Bayes factor ≥ 5 or p < 0.05.
Assembly: For splicing analysis, transcriptome annotation for the hg19/GRCh37 human genome assembly was created by merging gene annotations from Ensembl v71.1, the UCSC knownGene track, and MISO v2.0 isoform annotations.
Assembly: For gene expession analysis, hg38 reference genome was used.
Supplementary files format and content: comma-separated values file including splicing analysis for CB CD34+ HSPCs including dPSI and PSI values.
Supplementary files format and content: comma-separated values file including splicing analysis for PB CD34+ HSPCs including dPSI and PSI values.
Supplementary files format and content: comma-separated values file including splicing analysis for PB CD34+ HSPCs (SMARTSeq v4 Ultra Low Input RNA Kit) including dPSI and PSI values.
Supplementary files format and content: comma-separated values file including normalised counts for CD34+ HSPCs.
Supplementary files format and content: comma-separated values file including paiwise comparison between SF3B1 mutant and AAVS1 control CD34+ HSPCs.
Supplementary files format and content: comma-separated values file including splicing analysis for K562 cells including dPSI and PSI values.
Supplementary files format and content: comma-separated values file including normalised counts for K562 cells.
Supplementary files format and content: comma-separated values file including paiwise comparison between SF3B1 mutant and wild-type K562 cells.
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| Submission date |
Apr 05, 2024 |
| Last update date |
May 29, 2024 |
| Contact name |
Martina Sarchi |
| E-mail(s) |
martina.sarchi@unipv.it, doulatov@uw.edu
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| Organization name |
University of Pavia
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| Department |
Dept. of Molecular Medicine
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| Street address |
Via Ferrata 9
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| City |
Pavia |
| ZIP/Postal code |
27100 |
| Country |
Italy |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE263299 |
Mis-splicing of mitotic regulators sensitizes SF3B1-mutated human HSCs to CHK1 inhibition [RNA-seq] |
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| Relations |
| BioSample |
SAMN40761797 |
| SRA |
SRX24170363 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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