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| Status |
Public on Feb 22, 2019 |
| Title |
scRNA-seq on PBMCs from patient CLL5 after 30 days of ibrutinib treatment |
| Sample type |
SRA |
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| Source name |
PBMCs_CLL patient after 30 days of ibrutinib treatment
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| Organism |
Homo sapiens |
| Characteristics |
donor_id: CLL5
donor_type: chronic lymphocytic leukemia patient
treatment_timepoint_days: 30
cell type: Peripheral blood mononuclear cells (PBMCs)
replicate: 1
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| Treatment protocol |
All patients were diagnosed and treated and diagnosed according to the revised guidelines of the International Workshop Chronic Lymphocytic Leukemia/National Cancer Institute. Patients were recruited through clinical trial NCT01804686, aimed at studying the long-term effect of PCI-32765 (ibrutinib) and reflect the clinical and biological heterogeneity of the disease. The study was approved by the ethics committees of the contributing institutions (Semmelweis University and Medical University of Vienna). Informed consent was obtained from all participants.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
An aliquot of the patient PBMCs was stained with DAPI for LIVE/DEAD discrimination and a maximum of 100.000 LIVE, doublet-excluded cells sorted in 1.5 ml tubes. Cells were pelleted by centrifuging for 5 min at 4 °C at 300 x g and resuspended in PBS with 0.04% BSA.
Single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics) according to the manufacturer’s protocol. In brief, up to 17.000 cells suspended in reverse transcription reagents, along with gel beads, were segregated into aqueous nanoliter-scale gel bead-in-emulsions (GEMs). The GEMs were then reverse transcribed in a T1000 Thermal cycler (Bid-Rad) programed at 53 °C for 45 min, 85 ºC for 5 min, and hold at 4 ºC. After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was then amplified with a T1000 Thermal cycler programed at 98 ºC for 3 min, 10 cycles of (98 ºC for 15 s, 67 ºC for 20 s, 72 ºC for 1 min), 72 ºC for 1 min, and hold at 4 ºC. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed and index adaptor ligated, with SPRIselect Reagent Kit (Beckman Coulter) with cleanup in between steps. Post-ligation product was amplified with a T1000 Thermal cycler programed at 98 ºC for 45 s, 10 cycles of (98 ºC for 20 s, 54 ºC for 30 s, 72 ºC for 20 s), 72 ºC for 1 min, and hold at 4 ºC. The sequencing-ready library was cleaned up with SPRIselect and sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq4000 platform and the 75-bp paired-end configuration.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
scRNA-seq on PBMCs from patient CLL5 after 30 days of ibrutinib treatment
scRNA-seq_CLL5_30d
LiveBulk_CLL5_30d_10xTK118s_S5_L008
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| Data processing |
Preprocessing was performed using Cell Ranger (version 2.0.0) provided by 10x Genomics. Raw sequencing files were demultiplexed using the Cell Ranger command mkfastq. Each sample was aligned to the human reference genome refdata-cellranger-GRCh38-1.2.0 using Cell Ranger count, and all samples were aggregated using Cell Ranger aggr without depth normalization.
Genome_build: hg38
Supplementary_files_format_and_content: Each csv file contains read counts for each sample for each regulatory element; MTX (Matrix Market file format)
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| Submission date |
Feb 22, 2018 |
| Last update date |
Feb 22, 2019 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE111014 |
Chromatin mapping and single-cell immune profiling defines the temporal dynamics of ibrutinib drug response in chronic lymphocytic leukemia [scRNA-seq] |
| GSE111015 |
Chromatin mapping and single-cell immune profiling defines the temporal dynamics of ibrutinib drug response in chronic lymphocytic leukemia |
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| Relations |
| BioSample |
SAMN08581378 |
| SRA |
SRX3734633 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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