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Sample GSM3096721 Query DataSets for GSM3096721
Status Public on Apr 17, 2018
Title L1_I1_LUM_F1
Sample type SRA
Source name Mammoplasty Reduction
Organism Homo sapiens
Characteristics Sex: female
tissue: Adult Human Breast Epithelium
location: Luminal
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.
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/┬Ál. 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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
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
Submission date Apr 13, 2018
Last update date Apr 17, 2018
Contact name Kai Kessenbrock
Phone 9498243424
Organization name UC Irvine
Department Biological Chemistry
Street address 839 Health Sciences Road
City Irvine
State/province CA
ZIP/Postal code 92617
Country USA
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
BioSample SAMN08936050
SRA SRX3941004

Supplementary file Size Download File type/resource
GSM3096721_Ind1_Lib1_F1_gene_FPKM_withID.txt.gz 111.2 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record
Processed data provided as supplementary file

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