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| Status |
Public on Apr 04, 2022 |
| Title |
Patients_multiome_atac |
| Sample type |
SRA |
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| Source name |
Forearm skin atac-seq
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Skin
selection marker: CD90+
patient id: dSSc
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
human samples: skin samples were obtained through punch biopsy (4 mm), 10 cm distal to the elbow and placed in saline containing tubes on ice and then transferred to cold FACS buffer (EDTA pH8.0 2mM, BSA 0.5% in PBS). Whole blood samples were collected at the time of skin biopsy, were placed in EDTA-containing tubes (Beckton Dickenson) on ice and diluted 1:1 with ice cold FACS buffer. Skin and blood samples immediately transported to the lab.
To generate gentle and efficient single-cell suspensions from human skin tissue, we used Miltenyi human whole skin dissociation kit (catalog num. 130-101-540), which combines mechanical dissociation with enzymatic degradation of the extracellular adhesion proteins. In short, first we cut the skin biopsy into small pieces (< 1mm) and transferred them into gentleMACS C Tube containing 435µL of Buffer and 65 µL of enzyme mix. Next, samples were incubated in a water bath at 37 °C for 1h. After incubation we diluted the samples by adding 0.5 mL of cold cell culture medium, tightly closed C Tube and attached it upside down onto the sleeve of the gentleMACS dissociator, and run the gentleMACS Program h_skin_01. After a short centrifugation step to collect the sample material at the tube bottom, we applied the cell suspension to a 70 µm pre-separation filter, placed on a 15 mL tube, and washed with ice cold FACS buffer. Then, we centrifuged the cell suspension at 300×g for 10 minutes at 4°C and aspirated the supernatant completely. Finally, after red blood cell lysis (Sigma) for 5min at 4°C, centrifugation at 300×g for 10 minutes at 4°C, and washing, we resuspended the cells for FACS staining and sorting.
Skin cell suspension was stained with the following antibodies: CD45 (PerCp/Cyanine, 3040285.5, Biolegend), CD90 (PE 328144, Biolegend). All FACS antibodies were used with a 1:100 dilution. Cells were bulk sorted on FACS Symphony S6, cell sorter (BD Biosciences) using 100 nozzle and flowrate of 2 for 3 hours and pooled together. 75.000 and 40000 cells were obtained equally from 9 healthy and 9 dSSc patients, respectively.
Sorted cells were processed according to 10x Genomics Chromium Single Cell Multiome ATAC+ Gene Expression low cell number protocol according to protocol number CG000365_DemonstratedProtocol_NucleiIsolation_ATAC_GEX_Sequencing_RevB used for cell lysis (0.1x lysis buffer and lysed for 7 min) to obtain intact nuclei. Single cell ATAC and RNA-seq libraries were prepared using the Chromium single cell multiome ATAC + gene expression platform (10x Genomics). Nuclei were prepared and counted to ensure quality and concentration. Nuclei were then transposed according to the manufacturer’s protocol. Transposed nuclei suspension was loaded onto Next GEM Chip J targeting 5000 nuclei and then ran on a Chromium Controller instrument to generate GEM emulsion (10x Genomics). Single-cell gene expression libraries, as well as single cell ATAC-seq libraries, were generated according to the manufacturer’s protocol using the Chromium Next GEM Single Cell multiome ATAC+ gene expression kit. Final libraries were quantified using NEBNext Library Quant Kit for Illumina (NEB) and high sensitivity D1000 TapeStation (Agilent). Each library was sequenced separately on a NovaSeq 6000 instrument using an SP 100 cycles reagent kit (Illumina), targeting 25,000 reads/nuclei for ATAC-seq and a minimum of 20,000 reads/nuclei for gene expression.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
bcl2fastq/2.15.0.4
cellranger-arc/2.0.0
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include ATAC peak read count values and mRNA molecule count values for each Sample
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| Submission date |
Feb 10, 2022 |
| Last update date |
Apr 04, 2022 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
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| Phone |
972-8-9343338
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| Organization name |
Weizmann Institute of Science
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| Department |
Immunology
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| Street address |
234 Herzl st.
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| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE195452 |
LGR5 expressing skin fibroblasts define a major hub perturbed in Systemic Sclerosis |
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| Relations |
| BioSample |
SAMN25828887 |
| SRA |
SRX14126876 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5884740_Patients_atac.tar.gz |
102.9 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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