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| Status |
Public on Jul 05, 2022 |
| Title |
CH04_2 scRNAseq + Genotyping |
| Sample type |
SRA |
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| Source name |
GCSF mobilized stem cells from patient
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD34+
tissue: Blood
disease state: Clonal hematopoiesis
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cryopreserved patient samples approved by the Institutional Review Board of Dana Farber Cancer Institute were thawed and stained using standard procedures (10 min, 4°C) with the surface antibody CD34-PE-Vio770 (clone AC136, Miltenyi Biotec) and DAPI (Sigma-Aldrich). Cells were then sorted for DAPI-negative, CD34+ and CD34-negative cells using BD Influx.
Methylome and whole-transcriptome library preparation and sequencing were performed as previously described. Briefly, genomic DNA (gDNA) and mRNA have been separated manually. A modified oligo-dT primer (5′-biotin-triethyleneglycol-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, where V is either A, C or G, and N is any base; IDT) was conjugated to streptavidin-coupled magnetic beads (Dynabeads, Life Technologies) according to the manufacturer’s instructions. To capture polyadenylated mRNA, we added the conjugated beads (10 μl) directly to the cell lysate and incubated them for 20 min at room temperature with mixing to prevent the beads from settling. The mRNA was then collected to the side of the well using a magnet, and the supernatant, containing the gDNA, was transferred to a fresh plate. Single-cell complementary DNA was amplified from the tubes containing the captured mRNA according to a variation of the Smart-Seq2 protocol using molecular crowding to increase sensitivity. After amplification and purification using 0.8X SPRI beads, 0.5ng cDNA was used for Nextera Tagmentation and library construction. Library quality and quantity were assessed using Agilent Bioanalyzer 2100 and Qubit, respectively. Genomic DNA present in the pooled supernatant and wash buffer from the mRNA isolation step was concentrated on 0.8X SPRI beads and eluted directly into the reaction mixtures for Msp1(+/- HaeIII) (Fermentas) enzymatic reaction (10mL final reaction) for 90 min at 37°C. Heat-inactivation was performed for 10 min at 70°C. Digested DNA was filled-in and A-tailed at the 3’ sticky ends in 8.5 mL final volume of 1X CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas). Reaction was supplemented with 1 mM dATP and 0.1 mM dCTP and 0.1 mM dGTP (NEB) and performed as follows in a thermocycler: 30°C for 25 min, 37°C for 25 min and heat-inactivation at 70°C for 10 min. Custom barcoded methylated adaptors (0.1 mM) were then ligated overnight at 16°C with the dA-tailed DNA fragments in the presence of 800 units of T4 DNA ligase (NEB) and 1 mM ATP (Roche) in a final volume of 11.5 mL of 1X CutSmart buffer. T4 DNA ligase heat-inactivation was performed at 70°C for 15 min the next day. Genomic DNA from 24 individual cells were pooled together according to their barcodes, giving, for a 96-well plate, 4 pools of 24 cells. Pooled genomic DNA was cleaned-up and concentrated using 1.8X SPRI beads (Agencourt AMPure XP - Beckman Coulter). Each pool was then sodium bisulfite converted (Fast Epitect Bisulfite, Qiagen) following manufacture recommendations or converted using EM-seq kit (New Englands Biolabs) following manufacture recommendations. Converted DNA was then amplified using primers containing Illumina i7 and i5 index. Following Illumina pooling guidelines, a different i7 index was used for every 24-cell pool, allowing multiplexing of 96 cells for sequencing on one Illumina HiSeq lane. Library enrichment was done using KAPA HiFi Uracil+ master mix (Kapa Biosystems) and the following PCR condition was used: 98°C for 45 secs; 6 cycles of: 98°C for 20 secs, 58°C for 30 secs, 72°C for 1 min; followed by 12 cycles of: 98°C for 20 secs, 65°C for 30 secs, 72°C for 1 min. PCR was terminated by an incubation at 72°C for 5 min. Enriched libraries were cleaned-up and concentrated using 1.3X SPRI beads. DNA fragments between 200 bp and 1 Kb were size-selected and recovered after resolving on a 3% NuSieve 3:1 agarose gel. Libraries molarity concentration calculation was obtained by measuring concentration of double stranded DNA (Qubit) and quantifying the average library size (bp) using an Agilent Bioanalyzer. Every 24-cells pool was mixed with the others pool in an equimolar ratio. All cells from a 96-well plate were sequenced as paired-end on HiSeq 2500 with 10% PhiX spike-in. Negative controls (empty wells with no cells) were used to control for non-specific amplification of the libraries. Single cell bisulfite-seq or enzymatic methyl-seq (reduced representation) and single-cell RNA-seq (full-length transcriptome profiling)
Single cell bisulfite-seq or enzymatic methyl-seq (reduced representation) and single-cell RNA-seq (full-length transcriptome profiling)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
polyA mRNA
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| Data processing |
scRNA data was aligned with STAR (v2.5.2) using twopassMode, gene level counts were generated using featureCounts (subread v1.5.2) with default parameters. The number of mutant and WT reads per cell was counted using a pileup at the mutation site (pySAM v0.8.2.1). Only reads with MAPQ > 40 and a specified minimum base quality at the mutation site were considered.
For scDNAme, each pool of 96 cells was first demultiplexed by Illumina i7 barcodes, resulting in four pools of 24 cells. Each pool of 24 cells was further demultiplexed by unique cell barcodes. Reads were assigned to a given cell if they matched 80% of the template adapters. Adapters and adapter reverse complements (6 bp) were trimmed from the raw sequence reads. After adapter removal, reads were trimmed by 5 bp from their 3’ end. We aligned trimmed reads to the hg38 human genome assembly using Bismark117 (v.0.14.5; parameters: -multicore 4 -X 1000 --un –ambiguous) running on bowtie2-2.2.8 aligner118. Bismark methylation extractor (--bedgraph --comprehensive) was used to determine the methylation state of each individual CpG. For downstream analyses, a site was considered methylated or unmethylated only if there was 90% agreement of the methylation state for all reads mapped to the site. Cells with coverage of at least 25,000 unique CpGs and bisulfite conversion rates of at least 99% were retained for downstream analyses.
Genome_build: hg38
Supplementary_files_format_and_content: Text files include counts overlapping with annotated genes for all cells. Excel and comma-separated values files contain the filename conversion key to match scRNA with scDNAme cells, the sequencing summary statistics for each cell, and genotype call.
Supplementary_files_format_and_content: all_CpG_meth.csv: CpG by cell matrix with methylation values for all detected CpGs
Supplementary_files_format_and_content: scRNA_filename_key_to_scDNAme.csv: Filename conversion key to match scRNA with scDNAme cells
Supplementary_files_format_and_content: scDNAme_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: scRNA_cell_metadata.xlsx: Summary statistics and metadata of all cells
Supplementary_files_format_and_content: gene_exp_mtx.txt: Gene count matrix from single-cell RNA-seq
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| Submission date |
Oct 24, 2020 |
| Last update date |
Jul 05, 2022 |
| Contact name |
Neville Dusaj |
| E-mail(s) |
ned2010@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Lab |
Landau Lab
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| Street address |
413 E 69th St.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE158067 |
Single-cell multi-omics in human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation. |
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| Relations |
| BioSample |
SAMN16535293 |
| Supplementary data files not provided |
| Processed data are available on Series record |
| Raw data not provided for this record |
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