 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 30, 2020 |
| Title |
ALL8 |
| Sample type |
SRA |
| |
|
| Source name |
Primary pre-B-ALL BM
|
| Organism |
Homo sapiens |
| Characteristics |
time point: d0
tissue: bone marrow
|
| Treatment protocol |
All cases received standard induction therapy according to the NOPHO ALL-2008 protocol, with prednisolone 60 mg/m2/day p.o. days 1-29, vincristine 2.0 mg/m2 i.v. days 1, 8, 15, 22, and 29, doxorubicin 40 mg/m2 i.v. days 1 and 22, and methotrexate i.t. days 1, 8, 15, and 29
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mononuclear cells (MNC) were extracted from fresh BM at diagnosis using Ficoll-Paque Plus (GE Healthcare, #17-1440-02). Bone marrow MNCs were also extracted from two patients (ALL10 and ALL12) during the induction therapy at day 15 after initiation of therapy. MNCs were viably frozen in 15% DMSO/40% FBS in RPMI in liquid nitrogen. Cells from primary BM samples were processed for scRNA-seq in the Finnish Functional Genomics Center, Turku, Finland, in 4 batches: 1) ALL3, 2) ALL1 3) ALL10 and ALL10-d15, and 4) ALL8, ALL9, ALL12, and ALL12-d15. Before applying the cells into the Chromium cartridge, their viability was checked using Trypan blue. PI-negative (live) cells were selected from sample ALL3 using FACS. Samples ALL1, ALL10, and ALL10-d15 were processed directly after thawing the MNC fraction without further processing. Excess dead cells were depleted from samples ALL8 and ALL9 using bead-based Dead Cell Removal Kit (#130-090-101, MACS miltenyi Biotech), increasing the percentage of viable cells from 43 % to 72 % and from 63 % to 78 %, respectively. For samples ALL12 and ALL12-d15, enrichment of leukemic cells was carried out by depleting non-B-cells using streptavidin-beads (BD Streptavidin Particles Plus, BD Biosciences, Franklin Lakes, NJ, USA) and biotinylated antibodies against human CD16 (clone 3G8), CD14 (HCD-14), CD11c (3.9), CD56 (HCD56), CD3 (UCHT1), and CD66 (G10F5) (Biolegend), all with final concentrations of 2 ug/ml, following the manufacturer's instructions and as previously described (Good et al. 2018 Nat Meth). Depletion efficiency was estimated by flow cytometry using CD3 (BV421, BD Biosciences, #56287, RRID:AB_27378607) and CD19 (Thermo Fisher Scientific, # 25-0199-41, RRID:AB_1582279) antibodies, with a viability dye (eBioscience, Fixable Viability Dye eFluor™ 506, #65-0866-14). Depletion decreased the proportion of T-cells (CD3+) from 30 to 2%, increased the proportion of B-cells (CD19+) from 23 to 50%, and increased the percentage of viable cells from 50 to 80% in a test BM sample.
Cells were combined with reverse transcriptase Master Mix and partitioned into Gel Bead-In EMulsions (GEMs) using 10X GemCode Technology, where the poly-A transcripts are barcoded with an Illumina R1 sequence, a 16 bp 10X barcode and a 10 bp Unique Molecular Identifier (UMI). 11-12 cycles of PCR was used to amplify the cDNA.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Data was aligned with Cell Ranger 3.0.2 to human reference (hg19) version 3.0.0 from 10x Genomics with default parameters.
Cells were filtered with Cell Ranger 3.0.2 following alignment.
Genome_build: hg19
Supplementary_files_format_and_content: barcode annotation: 10x Cell Ranger filtered barcode list; feature annotation: 10x Cell Ranger feature list; count matrix: 10x Cell Ranger filtered UMI count matrix
|
| |
|
| Submission date |
Apr 07, 2020 |
| Last update date |
Oct 30, 2020 |
| Contact name |
Merja Heinäniemi |
| E-mail(s) |
merja.heinaniemi@uef.fi
|
| Organization name |
University of Eastern Finland
|
| Department |
School of Medicine
|
| Street address |
Yliopistonranta 1 E
|
| City |
Kuopio |
| ZIP/Postal code |
70211 |
| Country |
Finland |
| |
|
| Platform ID |
GPL21290 |
| Series (1) |
| GSE148218 |
Single cell characterization of arrested B-lymphoid differentiation and leukemic cell states in ETV6-RUNX1-positive pediatric leukemia |
|
| Relations |
| BioSample |
SAMN14548863 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4456253_ALL8_barcodes.tsv.gz |
21.0 Kb |
(ftp)(http) |
TSV |
| GSM4456253_ALL8_features.tsv.gz |
302.7 Kb |
(ftp)(http) |
TSV |
| GSM4456253_ALL8_matrix.mtx.gz |
21.6 Mb |
(ftp)(http) |
MTX |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
|
|
|
|
 |
