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Sample GSM4458369 Query DataSets for GSM4458369
Status Public on Oct 30, 2020
Title ALL13
Sample type SRA
Source name freshly isolated primary bone marrow cells
Organism Homo sapiens
Characteristics tissue: freshly isolated primary bone marrow cells
disease state: acute lymphoblastic leukemia
subtype: ETV6-RUNX1
cell type: ALL cells
Growth protocol Cells were freshly isolated from bone marrow samples collected at diagnosis using Ficoll separation of mononuclear cells.
Extracted molecule total RNA
Extraction protocol GRO-seq: Mononuclear cells (MNC) were extracted from fresh BM at diagnosis using Ficoll-Paque Plus (GE Healthcare, #17-1440-02). Nuclei were extracted from 20 million cells and frozen in -80C.
GRO-seq: Nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. Trizol was added RNA was extracted , DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP ab using magnetic bead separation and precipitated overnight. Poly(A)-tailing was followed by cDNA synthesis using complementary poly(T)-primers. Excess oligo was removed by Exonuclease I and cDNA fragments of were purified. The recovered cDNA was circularized, linearized, amplified for 13-14 cycles. The final product was ran on 10% TBE gel, gel purified and cleaned-up using ChIP DNA clean & Concentrator Kit. The final libraries were sequenced using an Illumina HiSeq 2000.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Description nascent RNA
Data processing Libraries were sequenced for 51 cycles (single-end) on an Illumina HiSeq 2000, according to the manufacturer’s instructions.
GRO-Seq reads were trimmed to remove A-stretches originating from the library preparation using the Homer software (homerTools trim) and from resulting sequences those shorter than 25 bp were discarded. Sequence reads were further quality filtered with the FASTX software (-q 10 -p 97)
GRO-seq reads were aligned to the hg19 genome assembly using Bowtie version 0.12.9 (-v 2 -k 3 -m 1 --best)
bigWig files were generated using the HOMER software
Genome_build: hg19
Supplementary_files_format_and_content: bigWig GRO-seq signal files, separately for plus and minus strand
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 GPL11154
Series (1)
GSE148218 Single cell characterization of arrested B-lymphoid differentiation and leukemic cell states in ETV6-RUNX1-positive pediatric leukemia
BioSample SAMN14549328

Supplementary file Size Download File type/resource
GSM4458369_ALL13_hg19neg.ucsc.bigWig 69.9 Mb (ftp)(http) BIGWIG
GSM4458369_ALL13_hg19pos.ucsc.bigWig 73.5 Mb (ftp)(http) BIGWIG
Raw data not provided for this record
Processed data provided as supplementary file

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