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| Status |
Public on Apr 02, 2021 |
| Title |
TEA-seq of leukapheresis-purified, 0.01% digitonin perm, FACS neutrophil-depleted PBMCs, method comparison, well 1 |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
donor_id: HMN200910
donor_source: BioIVT
purification_type: leukapheresis
preparation_type: permeabilized cells
preparation_conditions: 0.01% digitonin
cleanup_type: FACS
cleanup_conditions: Dead Cell/Debris/Neutrophil Depletion
library_type: TEA-seq
library_type: TEA-seq
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| Extracted molecule |
total RNA |
| Extraction protocol |
Biological specimens were purchased from BioIVT as cryopreserved PBMCs. All sample collections were conducted by BioIVT under IRB-approved protocols, and all donors sign informed consent forms. PBMCs sourced from BioIVT were isolated using leukapheresis. Following isolation, PBMCs were subjected to RBC lysis, washing, and counting. PBMC aliquots were cryopreserved in Cryostor CS10 (StemCell Technologies, 07930) and stored in vapor phase liquid nitrogen.
Cryopreserved PBMCs were thawed, stained for FACS, depleted of neutrophils, dead cells, and debris through FACS, stained with TotalSeq-A antibodies, and permeabilized using 0.01% digitonin. ATAC and Gene Expression libraries were prepared according to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338 Rev A) with several modifications. To generate a high-quality reference dataset, we generated four 10x Genomics wells of TEA-seq. For each well, transposition was performed by diluting 15,400 permeabilized cells to a final volume of 5 µl in isotonic Tagmentation Buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2, RNase Inhibitor 1U/µl (Lucigen NxGen, F83923-1)). Cells were mixed with 10 µl of a transposition master mix consisting of 7 µl ATAC Buffer B (10x Genomics, 2000193) and 3 µl ATAC Enzyme B (Tn5 transposase; 10x Genomics, 2000265) per reaction. Transposition was performed by incubating the prepared reactions on a C1000 Touch thermal cycler with 96–Deep Well Reaction Module (Bio-Rad, 1851197) at 37°C for 60 minutes, followed by a brief hold at 4°C. A Chromium NextGEM Chip J (10x Genomics, 2000264) was placed in a Chromium Next GEM Secondary Holder (10x Genomics, 3000332) and 50% Glycerol (Teknova, G1798) was dispensed into all unused wells. A master mix composed of Barcoding Reagent Mix (10x Genomics, 2000267), Reducing Agent B (10x Genomics, 2000087), Template Switch Oligo (10x Genomics, 3000228), and Barcoding Enzyme Mix (10x Genomics, 2000266) was then added to each sample well, pipette-mixed, and loaded into row 1 of the chip. Chromium Single Cell Multiome Gel Beads v1.1 (10x Genomics, 2000261) were vortexed for 30 seconds and loaded into row 2 of the chip, along with Partitioning Oil (10x Genomics, 2000190) in row 3. A 10x Gasket (10x Genomics, 3000072) was placed over the chip and attached to the Secondary Holder. The chip was loaded into a Chromium Single Cell Controller instrument (10x Genomics, 120270) for GEM generation. At the completion of the run, GEMs were collected, and reverse transcription and barcoding were performed by incubating GEMs on a C1000 Touch thermal cycler with 96–Deep Well Reaction Module at 37°C for 45 min, 25°C for 30 min, followed by a brief hold at 4°C. Upon completion of the GEM incubation 5 µl of Quenching Agent (10x Genomics, 2000269) was immediately added and mixed with the reaction solution. GEMs were separated into a biphasic mixture through addition of Recovery Agent (10x Genomics, 220016), the aqueous phase was retained and cleared of barcoding reagents using Dynabead MyOne SILANE (10x Genomics, 2000048) and 2.0x bead:sample ratio SPRIselect reagent (Beckman Coulter, B23318) cleanups, incubating for 10 minutes after each bead addition step. Barcoded ATAC and cDNA fragments were amplified using a PCR reaction consisting of 45 µl of template, 50 µl of Pre-Amp Mix (10x Genomics, 2000270), 5 µl of Pre-Amp Primers (10x Genomics, 2000271), and 1 µl of ADT-Rev-AMP (0.2 µM Supplementary File 5). Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 72°C for 5 min, 98°C for 3 min, for 7 cycles of: 98°C for 20 sec, 63°C for 30 sec, 72°C for 1 min, with a final extension of 72°C for 1 min. Amplified fragments were purified using a 2.0x bead:sample ratio SPRIselect reagent (Beckman Coulter, B23318) bead clean-up, incubating for 10 minutes after bead addition. Sequencing ready ATAC libraries were constructed by amplifying barcoded ATAC fragments in a sample indexing PCR consisting of SI-PCR Primer B (10x Genomics, 2000128), Amp Mix (10x Genomics, 2000047) and Sample Index Plate N, Set A (10x Genomics, 3000427) as described in the 10x Multiome User Guide. Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 45 sec, then 9 cycles of: 98°C for 20 sec, 67°C for 30 sec, 72°C for 20 sec, with a final extension of 72°C for 1 min. Final ATAC libraries were prepared using a dual-sided 0.6x/1.6x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. cDNA fragments were amplified using a PCR reaction consisting of 35 µl of template, 50 µl of Amp Mix (10x Genomics, 2000047), 15 µl of cDNA Primers (10x Genomics, 2000089), and 1 µl of ADT-Rev-AMP (2 µM Supplementary File 5). Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 3 min, then 8 cycles of: 98°C for 15 sec, 63°C for 20 sec, 72°C for 1 min, with a final extension of 72°C for 1 min. cDNA and ADT fragments were separated using a dual-sided SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. Large cDNA fragments were retained in an initial 0.6x bead:sample SPRIselect incubation, reactions were incubated on a magnet, and small unbound fragments containing ADTs were transferred to new wells where they were subjected to an additional 2.0x bead:sample SPRIselect cleanup. ADT fragments were amplified in a 15 cycle indexing PCR, substituting SI-PCR-Oligo primer (10 µM Supplementary File 5) for SI-P5-22. Final ADT libraries were prepared using a dual-sided 1.6x bead:sample SPRIselect reagent (Beckman Coulter, B23318) clean-up. cDNA Fragmentation, End-Repair, and A-tailing were performed in a joint reaction containing 25% of cDNA sample as described in the 10x Multiome User Guide. Briefly, cDNA was diluted in Buffer EB (Qiagen, 1014609), and combined with master mix of Fragmentation Buffer (10x Genomics, 2000091) and Fragmentation Enzyme (10x Genomics, 2000090). The reaction was incubated in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 4°C start, 32°C for 5 min, 65°C for 30 min, followed by a brief hold at 4°C. Reactions were purified using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up. Ligation was performed by mixing each sample with a master mix consisting of Ligation Buffer (10x Genomics, 2000092), DNA Ligase (10x Genomics, 220110), and Adapter Oligos (10x Genomics, 2000094), and incubating in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module for 15 minutes at 20°C, followed by a brief hold at 4°C. Ligation reactions were purified using a 0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) clean-up. Sequencing ready Gene Expression libraries were constructed by amplifying cDNA fragments in a sample indexing PCR consisting of Amp Mix (10x Genomics, 2000047) and Dual Index TT Set A (10x Genomics, 3000431) as described in the 10x Multiome User Guide. Amplification was performed in a C1000 Touch thermal cycler with 96–Deep Well Reaction Module: 98°C for 45 sec, for 14 cycles of: 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec, with a final extension of 72°C for 1 min. Final Gene Expression libraries were prepared using a dual-sided 0.6x/0.8x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5
X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz
X066-MP0C1W3_leukopak_perm-cells_tea_48M_adt_counts.csv.gz
X066-MP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: TEA-seq
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| Data processing |
Demultiplexing of raw base call files into FASTQ files was performed using bcl2fastq2 (Illumina v2.20.0.422, parameters: --create-fastq-for-index-reads, --minimum-trimmed-read-length=8, --mask-short-adapter-reads=8, --ignore-missing-positions, -ignore-missing-filter, --ignore-missing-bcls, -r 24 -w 24 -p 80). The --use-bases-mask option was used to adjust the read lengths for each library type as follows: Y28n*,I10,I10n*,Y90n* for Gene Expression, Y50n*,I8n*,Y16,Y50n* for ATAC, and Y28n*,I8n*,n*,Y90n* for ADT.
For analysis of TEA-seq data alone, the full available sequencing depth from each library for each well was used for alignment. To perform comparisons between methodologies, all scRNA-seq and scATAC-seq libraries were downsampled to 200 million FASTQ entries (200M of each sequenced read; ~20,000 reads per expected cell recovered at ~10,000 cell expected recovery), and all ADT libraries were downsampled to 48M reads. RNA and ATAC libraries were aligned using cellranger-arc software (v1.0.0, 10x Genomics) against 10x genomics reference refdata-cellranger-arc-GRCh38-2020-A using default parameters. ADT barcode counting was performed using BarCounter.
genome_build: Ensembl GRCh38-3.0.0
processed_data_files_format_and_content: Fragment TSVs (Tab separated), Single cell CSVs (Commas separated), and HDF5 file (Hierarchical Data Format).
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| Submission date |
Mar 02, 2021 |
| Last update date |
Apr 02, 2021 |
| Contact name |
Allen Institute For Immunology |
| E-mail(s) |
xiaojun.li@alleninstitute.org
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| Phone |
2065487135
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| Organization name |
Allen Institute
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| Street address |
615 Westlake Ave N, Seattle, WA
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE158013 |
Simultaneous trimodal single cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq |
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| Relations |
| BioSample |
SAMN18105813 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5123951_X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_fragments.tsv.gz |
427.2 Mb |
(ftp)(http) |
TSV |
| GSM5123951_X066-MP0C1W3_leukopak_perm-cells_tea_200M_atac_filtered_metadata.csv.gz |
671.9 Kb |
(ftp)(http) |
CSV |
| GSM5123951_X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_filtered_feature_bc_matrix.h5 |
61.6 Mb |
(ftp)(http) |
H5 |
| GSM5123951_X066-MP0C1W3_leukopak_perm-cells_tea_200M_cellranger-arc_per_barcode_metrics.csv.gz |
21.4 Mb |
(ftp)(http) |
CSV |
| GSM5123951_X066-MP0C1W3_leukopak_perm-cells_tea_48M_adt_counts.csv.gz |
10.1 Mb |
(ftp)(http) |
CSV |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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