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| Status |
Public on Apr 13, 2022 |
| Title |
AML01 negative control well 1 [AML01_neg_A01_181120_scRNAseq_S97] |
| Sample type |
SRA |
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| Source name |
negative control
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| Organism |
blank sample |
| Characteristics |
sample type: negative control
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| Treatment protocol |
Cell lines were treated with 100nM decitabine every 24 hours (0, 24 and 48 hours) and harvested at 72 hours
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| Growth protocol |
AML01: Enriched mononuclear cells were purified from peripheral blood using Lymphoprep density gradient medium (StemCell, catalog # 7851) and SepMate tubes (StemCell, catalog # 85450), and cryopreserved. Cryopreserved primary human cells were resuspended in thawing media (IMDM, 20% FBS), washed twice and resuspended in thawing media. The cells were then rested for 1h at 37°C before preparation for flow cytometry. Cells (1x106/100ul) were stained with 1.5μg/mL propidium iodide (PI, Sigma-Aldrich), 1:20 CD45-PECy7 (2D1, Invitrogen), 1:20 CD33-FITC (WM-53, Invitrogen) and 1:20 CD19-BV711 (SJ25C1, BD Biosciences). Single blasts (PI-CD45dim) were collected in 2.5μL RLT Plus Lysis Buffer containing 1U/μL SUPERase-In (Invitrogen) in 96 well plates.
KG1a: KG1a cells were resuspended in binding buffer before staining with annexin V and 7-AAD. Live cells (Annexin V - /7-AAD -) were sorted into individual wells of a 96 well plate containing lysis buffer 2.5μL RLT Plus Lysis Buffer (QIAGEN, catalog # 1053393) containing 1U/μL SUPERase-In (ThermoFisher, catalog # AM2696) in 96 well plates. Before sorting, bulk samples of 1000000 cells were collected from both the untreated and treated populations.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
We utilised the G&T-seq protocol to separate genomic DNA and RNA from the single-cell samples. [Macaulay, I.C., et al., G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 2015]
Matched single cell RNA sequencing (scRNA-seq) libraries were prepared as per the scM&T-seq protocol. [Macaulay, I.C., et al., G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 2015]
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file: AML01_scRNAseq_TEtranscripts_counts.txt.gz
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| Data processing |
scRNA-seq data was trimmed using Trim Galore (v0.6.5), with default setting in paired-end mode.
Hisat2 (v2.1.0) and Samtools (v1.1.0) were used to convert, map and align unique and ambiguous reads to the human reference genome build GRCh38 from raw fastq reads into bam format.
TEtranscripts [Jin, Y., et al., TEtranscripts: a package for including transposable elements in differential expression analysis of RNA-seq datasets. Bioinformatics, 2015] was used to obtain raw gene and transposable element counts from the unique and ambiguously aligned reads using the GTF files for 1) TEs (http://labshare.cshl.edu/shares/mhammelllab/www-data/TEtranscripts/TE_GTF/) and 2) genes (https://asia.ensembl.org/info/data/index.html; release 101 from the FTP server) in GRCh38 ensembl format. TEtranscripts was run in a Conda (Inc., A., Anaconda Software Distribution. 2020, Anaconda Inc.) environment setup with Python (v3.7.7), Pysam (v0.16.0.1), R-base (v4.0.3) and Bioconductor-Deseq2 (v1.28.0).
Genome_build: GRCh38 ensembl format
Supplementary_files_format_and_content: The read count table are in tab-deliminated format with the first column containing the gene ensembl ID and TE name, followed by each column containing an individual named sample and associated counts.
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| Submission date |
Mar 29, 2021 |
| Last update date |
Apr 13, 2022 |
| Contact name |
Heather Lee |
| E-mail(s) |
Heather.lee@newcastle.edu.au
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| Organization name |
The University of Newcastle
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| Street address |
University Drive, Callaghan
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| City |
Newcastle |
| State/province |
NSW |
| ZIP/Postal code |
2308 |
| Country |
Australia |
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| Platform ID |
GPL29107 |
| Series (2) |
| GSE171027 |
SINEultaneous profiling of epigenetic heterogeneity and transcriptome in single cells [RNA-seq] |
| GSE171029 |
SINEultaneous profiling of epigenetic heterogeneity and transcriptome in single cells |
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| Relations |
| BioSample |
SAMN18524638 |
| SRA |
SRX10465663 |
| 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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