 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 13, 2022 |
| Title |
AML01_Alu_pool_cell_88 [i508_H4_i704_201027_AML01_Alu_pool_S4_L001] |
| Sample type |
SRA |
| |
|
| Source name |
patient sample
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: untreated
cancer type: Acute myeloid leukemia
Sex: M
age: 60
sample type: genomic DNA, bisulfate treated
|
| Treatment protocol |
NA
|
| Growth protocol |
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.
|
| Extracted molecule |
genomic DNA |
| 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.]. Genomic DNA from each cell was purified and bisulphite conversion was performed as described [Angermueller, C., et al., Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity. Nature methods, 2016], with minor modifications.
Bisulphite conversion was carried out using the EZ-96 DNA Methylation-Direct MagPrep Kit (Integrated Sciences, catalog # D5054) with half volumes of the manufacturer’s instructions. Bisulphite converted DNA was eluted directly from MagBeads into PCR-mix, and amplification of TEs was performed with MagBeads still in the well. PCR cycling conditions used were 95°C for 5 min (1 cycle), 98°C for 20 sec, 53°C for 15 sec, 72°C for 1 min (35 cycles), and 72°C for 10min (1 cycle). PCR mix used 7.5µl 1x KAPA HiFi hotStart Uracil + ReadyMix (Millennium, catalog # ROC-07959079001) and 0.3µM primer mix. Primers were designed against SINE Alu and LINE-1 consensus sequences (Supplementary Figure S2A) and are contained in Supplementary Tables S1 and S2. After amplification, plates of 96 single cell libraries purified using a 1.2x volume of AMPure XP beads (Beckman Coulter, catalog # A63881). All libraries were then quantified using the Qubit dsDNA HS kit (Life Technologies), normalised and pooled to a single tube. Pools where then added to 0.8µM NEBNext dual index oligo sets (Genesearch, catalog # E7780S) and 14.5µl 1x KAPA HiFi HotStart ReadyMix (Millennium, catalog # ROC-07958935001) for indexing and adaptor addition. PCR cycling conditions used were 98°C for 45 sec (1 cycle), 98°C for 15 sec, 65°C for 30 sec, 72°C for 30 sec (5 cycles), and 72°C for 5 min (1 cycle). Pools were then purified using 0.9x volume of Ampure XP beads, normalised and combined for sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
library strategy: single-cell transposable element methylation sequencing (scTEM-seq)
After initial demultiplexing of primary Illumina indexes, Cutadapt (v2.10) [18] was used to demultiplex pools based on custom secondary indexes
Commands –g and -G were used to pass named forward and reverse index lists as a .fasta file to Cutadapt for demultiplexing
Bisulfite reads were trimmed using Trim Galore (v0.6.5). 10bp was trimmed from both the 5’ and 3’ ends to remove oligo sequences from reads.
Reads were mapped to Bowtie2 (v 2.4.1) indexed human genome (GRCh38) non-directionally and as paired end using Bismark (v0.22.3)
The methylation extraction module from Bismark was then used to produce coverage files (.cov) for methylation analysis. We excluded cells with coverage of less than 1000 annotated TE sites.
Downstream analysis was performed using Seqmonk and custom R scripts
Genome_build: GRCh38
Supplementary_files_format_and_content: The genome-wide CpG methylation reports (.cov files) are tab-delimited, use 1-based genomic coordinates and are in the following format: <chromosome> <position> <methylation rate>
|
| |
|
| Submission date |
Mar 29, 2021 |
| Last update date |
Apr 13, 2022 |
| Contact name |
Heather Lee |
| E-mail(s) |
Heather.lee@newcastle.edu.au
|
| Organization name |
The University of Newcastle
|
| Street address |
University Drive, Callaghan
|
| City |
Newcastle |
| State/province |
NSW |
| ZIP/Postal code |
2308 |
| Country |
Australia |
| |
|
| Platform ID |
GPL15520 |
| Series (2) |
| GSE171026 |
SINEultaneous profiling of epigenetic heterogeneity and transcriptome in single cells [AML01 scTEM-seq] |
| GSE171029 |
SINEultaneous profiling of epigenetic heterogeneity and transcriptome in single cells |
|
| Relations |
| BioSample |
SAMN18524756 |
| SRA |
SRX10465606 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5216728_i508_H4_i704_201027_AML01_Alu_pool_CpG-meth_processed.cov.gz |
128.7 Kb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
