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| Status |
Public on Apr 02, 2021 |
| Title |
ICICLE-seq of leukapheresis-purified, 0.01% digitonin permeabilized PBMCs, well 3 |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
donor_id: HMN85396
donor_source: BioIVT
purification_type: leukapheresis
preparation_type: permeabilized cells
preparation_conditions: 0.01% digitonin
cleanup_type: None
cleanup_conditions: None
library_type: ICICLE-seq
library_type: ICICLE-seq
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| Extracted molecule |
genomic DNA |
| 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.
PBMCs were depleted of neutrophils, dead cells, and debris through FACS. 2×106 sorted PBMCs were centrifuged (400×g for 5 minutes at 4°C) using a swinging bucket rotor (Beckman Coulter Avanti J-15RIVD with JS4.750 swinging bucket, B99516), the supernatant was removed using a vacuum aspirator pipette, and the cell pellet was resuspended in 100 µl of DPBS without calcium and magnesium (Corning 21-031-CM) supplemented with 2% w/v BSA (Sigma-Aldrich A2934). 10 µl TruStain FcX (BioLegend, 422302) was added and cells were incubated on ice for 10 minutes. A panel of 46 barcoded oligo-conjugated antibodies (BioLegend TotalSeq-A) including a mouse IgG1? isotype negative control (Supplementary File 6) was added and incubated on ice for 30 minutes. Cells were washed three times in 4 mL of DPBS plus 2% BSA to remove unbound antibodies and used as input into cell permeabilization with 0.01% digitonin. Transposition was performed by aliquoting 20,000 permeabilized cells in TD1 buffer (Illumina, 15027866), bringing the volume up to 9 µl in TD1 buffer, and mixing with 6 µl of Poly-T overhang Tn5 complexes. Reactions were incubated on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module (Bio-Rad, 1851197) at 37°C for 120 minutes, followed by a brief hold at 4°C. Cell barcodes were then added to ATAC and antibody derived tags (ADTs) via GEM generation using 10x Genomics 3’ RNA beads and subsequent amplification. Briefly, a Chromium Next GEM Chip G (10x Genomics, 2000177) was placed in a Chromium Next GEM Secondary Holder (10x Genomics, 3000332) and 50% Glycerol (Teknova, G1798) was dispensed into all unused wells. A barcoding master mix was prepared which consisted of NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544), Reducing Agent B (10x Genomics, 2000087), F BC Primer (0.2 µM Supplementary File 5), and ADT-Rev-AMP (0.2 µM Supplementary File 5). The master mix was added to each 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 (10x Genomics, 120270) for GEM generation. At the completion of the run, GEMs were collected and amplification was performed on a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 72°C for 5 min, 98°C for 30 sec, 12 cycles of: 98°C for 10 sec, 42°C for 30 sec and 65°C for 30 sec, followed by a final extension of 65°C for 1 min. 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 (10x Genomics, 2000048) beads. Next, a dual-sided 0.6x/2.0x bead:sample SPRIselect reagent (Beckman Coulter, B23318) size-selection clean-up was performed to remove large DNA fragments and unused primers. Libraries were split into two reactions in a 3:1 ATAC:ADT ratio and amplified separately using different indexed P7 primers. ATAC fragments were amplified in a 100 µl reaction consisting of Buffer EB (Qiagen, 1014609), Amp Mix (10x Genomics, 2000047), SI-P5-22 primer (20 µM Supplementary File 5), and Chromium i7 Multiplex Kit N Set A (10x Genomics, 3000262). ATAC PCR was performed in a C1000 Touch thermal cycler with 96-Deep Well Reaction Module: 98°C for 45 sec, 7 cycles of: 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec, followed by a final extension of 72°C for 1 min. ADT fragments were amplified in a 100 µl reaction consisting of Buffer EB (Qiagen, 1014609), KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KM2602), SI-P5-22 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, 15 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 1.2x bead:sample ratio for ADT libraries and a dual-sided size-selection of 0.4x/1.2x bead:sample ratio for ATAC libraries. Final libraries were quantified using qPCR (KAPA Biosystems Library Quantification Kit for Illumina, KK4844) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, 1855195). Library quality and average fragment size were assessed using a Bioanalyzer (Agilent, G2939A) High Sensitivity DNA chip (Agilent, 5067-4626). Libraries were sequenced on the Illumina NovaSeq platform with the following read lengths: 28 bp read 1 (Cell barcode and UMI), 8 bp i7 index, 100 bp read 2 (ATAC-seq sequence or ADT barcode). A Truseq read 1 primer (0.3 µM Supplementary File 5) was included as a Custom Read 1 primer to mitigate the risk of off-target priming of the standard Illumina Nextera read 1 primer on the partial Nextera R1N sequence included in the mosaic end portion of the Poly-T Tn5 insertion.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-fragments.tsv.gz
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-metadata.csv.gz
X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_20kb-window-matrix.h5
X044-AP0C1W3
The FASTQs are from human subjects and will be provided via dbGAP
OTHER: ICICLE-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).
Read 2 was trimmed of adapter sequences, low quality bases and reads, and polyA tailing using fastp (S. Chen et al., 2018) v0.21.0 (parameters: --adapter_sequence=CTGTCTCTTATACACATCT --cut_tail --trim_poly_x).
Trimmed read 2 sequences were aligned to the GRCh38 (hg38) reference genome (Illumina iGenomes, https://support.illumina.com/sequencing/sequencing_software/igenome.html) using Bowtie 2 (Langmead and Salzberg, 2012; v2.3.0, parameters: --local, --sensitive, --no-unal, --phred33).
Aligned reads in SAM format were filtered by alignment score (greater than or equal to 30) then tagged with cell barcode and UMI sequence and quality scores using custom python code (python3 v3.7.3). Barcode sequences were compared against the 10x Genomics v3 3’ GEX barcode whitelist (3M-february-2018.txt.gz). Sequences not included in the whitelist were corrected to a valid whitelist barcode by allowing a single base mismatch (Hamming distance of 1). Sequences with more than one possible match were corrected at the position with the lowest sequencing quality score. Reads with barcodes that could not be corrected were excluded from further analysis. Filtered and tagged SAM files were converted to sorted, indexed BAM files using GATK (McKenna et al., 2010; Broad Institute v4.1.4.0). Genomic coordinates were converted to BED format using bedtools (Quinlan and Hall, 2010; v2.26.0). Custom python code was used to collapse aligned fragments into a list of fragments with unique cell barcode and genomic coordinate combinations. These fragments were then written as a fragments.tsv.gz file in the format: chr, start position, end position, cell barcode, UMI count, and strand (+/-).
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 |
Sep 15, 2020 |
| 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 |
SAMN16170663 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4784088_X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_20kb-window-matrix.h5 |
7.3 Mb |
(ftp)(http) |
H5 |
| GSM4784088_X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-fragments.tsv.gz |
33.6 Mb |
(ftp)(http) |
TSV |
| GSM4784088_X044-AP0C1W3_leukopak_perm-cells_icicle_fulldepth_filtered-metadata.csv.gz |
434.9 Kb |
(ftp)(http) |
CSV |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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