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Sample GSM4456254 Query DataSets for GSM4456254
Status Public on Oct 30, 2020
Title ALL9
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 eFluorâ„¢ 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
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
BioSample SAMN14548862

Supplementary file Size Download File type/resource
GSM4456254_ALL9_barcodes.tsv.gz 38.0 Kb (ftp)(http) TSV
GSM4456254_ALL9_features.tsv.gz 302.7 Kb (ftp)(http) TSV
GSM4456254_ALL9_matrix.mtx.gz 47.5 Mb (ftp)(http) MTX
Processed data provided as supplementary file
Raw data not provided for this record

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