GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1649153

Query DataSets for GSM1649153

Status

Public on Jul 05, 2016

Title

Nalm6 cell line [Gro-Seq]

Sample type

SRA

Source name

Nalm6 cell line

Organism

Homo sapiens

Characteristics

cell line: ALL leukemia cell line Nalm6
culture: alone
time: 0 h
replicate: 1

Treatment protocol

Cells in basal conditions

Growth protocol

Cells were cultured in RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) suppelemented with 2 mM L-glut, 100 U penicillin, 100 ug/ml streptomycin and 10% Tet System Approved FBS (Clontech, Mountain View, CA, USA). Co-cultured cells were grown separated by membrane with human primary stromal cells isolated from bone marrow.

Extracted molecule

total RNA

Extraction protocol

GRO-Seq: Nuclei were extracted from 5-16 million cells and nuclear run-on reactions were performed for 5 minutes at 30°C in the presence of Sarkosyl and BrUTP. RNA was extracted with Trizol, DNase-treated, base-hydrolyzed and dephosphorylated with PNK. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads 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 11-14 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.
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 11-14 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

Sample name: Nalm6_a
Nalm6 basal
nascent RNA
Nalms_minus_1e7_fs1e50_res50.bedGraph.gz, Nalms_plus_1e7_fs1e50_res50.bedGraph.gz
GRO-seq

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)
Bedgraphs were generated using the HOMER software from combined tagDirectories (per cell line). Data analysis was performed using HOMER and custom scripts http://biowhat.ucsd.edu/homer/
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph GRO-seq signal files, separately for plus and minus strand

Submission date

Apr 02, 2015

Last update date

May 15, 2019

Contact name

Merja Heinäniemi

Organization name

university of eastern finland

Street address

Yliopistonranta 1

City

Kuopio