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Sample GSM5837263 Query DataSets for GSM5837263
Status Public on Apr 04, 2022
Title AB7077
Sample type SRA
 
Source name Forearm skin
Organism Homo sapiens
Characteristics tissue: Skin
selection marker: CD45+
patient id: pt01002
Treatment protocol treatment protocol (1): 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.
treatment protocol (2): Blood mononuclear cell separation was performed by density centrifugation media (Ficol-paque, GE Life Sciences) in a 1:1 ratio with blood cells. Centrifugation (460g, 25min) was performed at 10°C, and the mononuclear cells were carefully aspirated and washed with ice cold FACS buffer. After red blood cell lysis (Sigma) for 5 minutes at 4°C, centrifugation at 300×g for 10 minutes at 4°C, and washing, we resuspended the cells for FACS staining and sorting.
Extracted molecule polyA RNA
Extraction protocol human sample collection: 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.
Skin cell suspension and blood mononuclear cells were stained with the following antibodies: CD45 (PerCp/Cyanine, 3040285.5, Biolegend), CD90 (PE 328144, Biolegend), LGR5 (PE, 373803, Biolegend), CD55 (FITC, 311306, Biolegend). All FACS antibodies were used with a 1:100 dilution. Samples were filtered through a 40μm strainer before commencing sorting. Single cell sorting was performed using FACSAria Fusion cell sorter (BD Biosciences, San Jose, CA). After doublets exclusion, isolated live cells were single-cell sorted into 384-well cell capture plates containing 2μL of lysis solution and barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq. Four empty wells were kept in each 384-well plate as a no-cell control for data analysis. Immediately after sorting, each plate was spun down to ensure cell immersion into the lysis solution, snap frozen on dry ice, and stored at –80°C until processed. Cells were analyzed using BD FACSDiva software (BD Bioscience) and FlowJo software (FlowJo, LLC).
Single-cell RNA-seq libraries were prepared as previously described {Jaitin, 2014}. In brief, mRNA from single cells sorted into capture plates were barcoded and converted into cDNA and then pooled using an automated pipeline. The pooled sample was linearly amplified by T7 in vitro transcription, and the resulting RNA was fragmented and converted into a sequencing-ready library by tagging the samples with pool barcodes and Illumina sequences during ligation, RT, and PCR. Each pool of cells was tested for library quality and concentration as described previously {Jaitin, 2014}
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing bcl2fastq/2.15.0.4
Sequences with RMT of low quality (defined as RMT with minimum Phred score of less than 27) were filtered out.
Well-barcode-RMT are extracted from the 9 first base pairs in the R2 fastq sequences. Unique-molecular-identifier are extracted from the subsequent 8 base pairs in the R2 sequences. R1 contains the genomic sequences.
Reads were separated by POOL_BARCODE_WELL_BARCODE header data, allowing 1 sequencing error. This process created a single fastq file for each source well.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include mRNA molecule count values for each Sample
 
Submission date Jan 26, 2022
Last update date Apr 04, 2022
Contact name Ido Amit
E-mail(s) ido.amit@weizmann.ac.il
Phone 972-8-9343338
Organization name Weizmann Institute of Science
Department Immunology
Street address 234 Herzl st.
City Rehovot
ZIP/Postal code 760001
Country Israel
 
Platform ID GPL18573
Series (1)
GSE195452 LGR5 expressing skin fibroblasts define a major hub perturbed in Systemic Sclerosis
Relations
BioSample SAMN25279335
SRA SRX13925572

Supplementary file Size Download File type/resource
GSM5837263_AB7077.txt.gz 618.1 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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