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| Status |
Public on Apr 02, 2021 |
| Title |
CITE-seq of leukapheresis-purified, 0.01% digitonin permeabilized, FACS neutrophil-depleted PBMCs, method comparison |
| 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: whole cells
preparation_conditions: N/A
cleanup_type: FACS
cleanup_conditions: Dead Cell/Debris/Neutrophil Depletion
library_type: CITE-seq
library_type: CITE-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.
CITE-seq library generation was performed using 10x Genomics Single Cell 3’ v3.1 kit, based on the 10x 3’ Gene Expression Library Construction Guide (CG000204 Rev B) and the New York Genome Center Technology Innovation Lab CITE-seq protocol (Version 2019-02-13). Briefly, a Chromium Next GEM Chip G (10x Genomics, 2000177) was placed in a Chromium Next GEM Secondary Holder and 50% Glycerol was dispensed into all unused wells. A barcoding master mix was prepared which consisted of RT Reagent B (10x Genomics, 2000165), Template Switch Oligo (10x Genomics, 3000228), Reducing Agent B (10x Genomics, 2000087), RT Enzyme C (10x Genomics, 2000085). The master mix was added to the sample well, pipette-mixed, and loaded into row 1 of the chip. Chromium Single Cell 3’ v3.1 Gel Beads (10x Genomics, 2000164) 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, 370017) was placed over the chip and attached to the Secondary Holder. The chip was loaded into a Chromium Single Cell Controller instrument for GEM generation. GEMs were separated into a biphasic mixture through addition of Recovery Agent (10x Genomics, 220016), the aqueous phase was retained and removed of barcoding reagents using Dynabead MyOne SILANE beads. cDNA amplification was performed using the 10x Genomics cDNA Primers (2000089) on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 53°C for 45 min, 85°C for 5 min, followed by a hold at 4°C. To separate ADT and cDNA libraries, we performed a 0.6x bead:sample SPRIselect reagent size-selection. The bead fraction from this separation was retained as the cDNA fraction, and supernatant separation was retained as the ADT-containing fraction. To further process ADTs, we added an additional 1.4x SPRI volumes to the ADT-containing supernatant to a final ratio of 2x SPRI:sample, then retained the bead fraction of this second step. We then repeated a fresh 2x bead:sample cleanup, retaining the bead fraction as the ADT library. ADTs were amplified in a 100 µl reaction consisting of Buffer EB, KAPA HiFi HotStart ReadyMix, SI-PCR-Oligo primer (10 µM Supplementary File 5), and ADT i7 primer (10 µM Supplementary File 5). ADT PCR was performed in a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 95°C for 3 min, 12 cycles of: 95°C for 20 sec, 60°C for 30 sec, 72°C for 20 sec, followed by a final extension of 72°C for 5 min. SPRIselect reagent cleanups were performed with a 1.6x bead:sample ratio to generate final ADT libraries.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 NextSeq 500 |
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| Description |
X066-RP0C1W1_leukopak_perm-cells_cite_200M_rna_counts.h5
X066-RP0C1W1_leukopak_perm-cells_cite_48M_adt_counts.csv.gz
X066-RP0C1W1
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: CITE-seq
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| Data processing |
Demultiplexing of CITE-seq raw base call files into FASTQ files was performed using 10x Genomics cellranger mkfastq (v.3.0.2).
To perform comparisons between these 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 (Limited by the available read count for 1 experiment; ~4,800 reads per expected cell recovered).
For CITE-seq data, RNA alignment was performed using cellranger (v5.0.0) with the –include-introns parameter against 10x Genomics reference refdata-gex-GRCh38-2020-A. To analyze CITE-seq ADT libraries we used BarCounter to compute the counts of ADT barcodes for each 10x cell barcode
genome_build: Ensembl GRCh38-3.0.0
processed_data_files_format_and_content: 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 |
GPL18573 |
| Series (1) |
| GSE158013 |
Simultaneous trimodal single cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq |
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| Relations |
| BioSample |
SAMN18105809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5123955_X066-RP0C1W1_leukopak_perm-cells_cite_200M_rna_counts.h5 |
29.9 Mb |
(ftp)(http) |
H5 |
| GSM5123955_X066-RP0C1W1_leukopak_perm-cells_cite_48M_adt_counts.csv.gz |
20.2 Mb |
(ftp)(http) |
CSV |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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