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| Status |
Public on Apr 17, 2018 |
| Title |
L1_I1_LUM_G10 |
| Sample type |
SRA |
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| Source name |
Mammoplasty Reduction
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| Organism |
Homo sapiens |
| Characteristics |
Sex: female
tissue: Adult Human Breast Epithelium
location: Luminal
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| Treatment protocol |
Samples were washed in PBS (Corning 21-031-CV) and mechanically dissociated using a razor blade. Dissociated samples were digested overnight in DMEM (Corning 10-013-CV) with Collagenase Type I, 2 mg/mL (Life Technologies 17100-017). Viable organoids were separated using differential centrifugation and viably frozen in 50% FBS (Omega Scientific FB-12), 40% DMEM, and 10% DMSO (Sigma-Aldrich D8418) by volume.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Viable organoids were thawed and washed using DMEM, and digested with 0.05% trypsin (Corning 25-052-CI) containing DNase (Sigma Aldrich D4263-5VL) to generate single cell suspension. Cells were stained for FACS using fluorescently labeled antibodies for CD31 (eBiosciences 48-0319-42), CD45 (eBiosciences 48-9459-42), EpCAM (eBiosciences 50-9326-42), CD49f (eBiosciences 12-0495-82), SytoxBlue (Life Technologies S34857). We only proceeded with samples showing at least 80% viability as measured using SytoxBlue in FACS
Sorted cells were washed and resuspended at a concentration of approximately 500 cells/µl. For microfluidics-enabled scRNAseq, cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100-5315) at a ratio of 8:2 before loading mix onto C1 chip (Fluidigm 100-5760). Bright field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol: 100-7168 I1. RNA spike-in controls were omitted. cDNA library preparation were performed following the Fluidigm C1 Protocol: 100-7168 I1. Reagents Kits v2 User Guide: CG00052 Rev B. Quantification of cDNA libraries was performed using Qubit dsDNA HS Assay Kit (Life Technologies Q32851) and high-sensitivity DNA chips (Agilent. 5067-4626). Quantification of library construction was performed using KAPA qPCR (Kapa Biosystems KK4824). For microfluidics-enabled scRNAseq libraries, we generally multiplexed 96 cells per lane on an Illumina HiSeq2500 resulting in a calculated depth of ~1.6 million reads per cell (Illumina Rapid PE kit v2 402-4002 and Rapid SBS kit v2 FC 401-4022).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
After demultiplexing sequencing libraries to individual cell FASTQ files (observed average read depth per cell was found to be ~1.6 Million reads), each library was aligned to an indexed GRCh38 RefSeq genome using RSEM version 1.2.1242, and bowtie2 version 2.2.3 with the following options enabled: rsem-calculate- expression -p $CORES -- bowtie2 -- paired-end -output- genome-bam. Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were quantified and concatenated into a resulting gene expression matrix for each library
Genome_build: GrCH38
Supplementary_files_format_and_content: Processed Data files are tab-delimited .txt files of counts expression matrices corresponding to each of the libraries generated from the 3 individuals in this study
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| Submission date |
Apr 13, 2018 |
| Last update date |
Apr 17, 2018 |
| Contact name |
Kai Kessenbrock |
| E-mail(s) |
kessenbrocklab@gmail.com
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| Phone |
9498243424
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| Organization name |
UC Irvine
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| Department |
Biological Chemistry
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| Street address |
839 Health Sciences Road
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| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92617 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE113099 |
Single Cell RNA sequencing of Adult Human Breast Epithelial Cells [C1_Individual_1] |
| GSE113197 |
Single Cell RNA sequencing of Adult Human Breast Epithelial Cells |
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| Relations |
| BioSample |
SAMN08936039 |
| SRA |
SRX3941025 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3096742_Ind1_Lib1_G10_gene_FPKM_withID.txt.gz |
112.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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