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| Status |
Public on Dec 07, 2020 |
| Title |
Patient#6_Carbo/DMSO |
| Sample type |
SRA |
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| Source name |
AB-33A (BY1146)
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| Organism |
Homo sapiens |
| Characteristics |
cell type: breast cancer tumor cell
treatment: Carboplatin (0.1mM)+DMSO
patient: 6
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| Treatment protocol |
Patient cells were counted and plated at 10,000 per well in 100 µL RETM with 96-well spheroid plates with black walls (Corning 4520). After 18-24 hours, 100 µL culture medium with drug (2X dose) was added. After 72 hours, the medium was removed, and the organoids were washed with fresh medium once and replaced with 200 µL fresh medium containing CSL inhibitors.
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| Growth protocol |
For single cell RNA sequencing, we thawed frozen viable breast cancer pleural effusions or ascites from patients and depleted dead cells with EasySepTM Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies) and CD45+ cells with EasySepTM Human CD45 Depletion Kit II (STEMCELL Technologies) according to manufacturer protocols. These purified cancer cells were then resuspended in Renaissance Essential Tumor Medium (RETM, Cellaria) containing 5% FBS, RETM supplement, antibiotic/antimycotic (anti-anti), and 20 ng/mL cholera toxin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell organoids were collected by centrifugation at 300g for 5min, and dissociated with trypsin-EDTA for 5min at 37°C. Dead cells were depleted with EasySepTM Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, #17899). Cells in suspension were stained with ReadyProbes Cell Viability Imaging Kit (Hoechst 33342 and Propidium Iodide, Thermo Fisher Scientific) by adding 80µL each dye at 500,000 cell/mL in basal DMEM medium and incubated for 20 minutes at 37°C + 5% CO2. After incubation, cells were diluted with equal volume of 1X PBS (no Ca2+/Mg2+, pH 7.4, Thermo Fisher Scientific) then centrifuged for 5 minutes at 300 x g, 4°C. Cells were resuspended to 5.0x105 to 2.0x106 cell/mL in 1X PBS and calculated concentrations on a hemocytometer using trypan blue then diluted to 50,000 cell/mL in 1x PBS + 0.2U/µL Superase· In RNase inhibitor (Thermo Fisher Scientific) + 1X Second Diluent (Takara Bio). Single-cell RNA-Sequencing (scRNA-Seq) was performed using the ICELL8® Single-Cell System (Takara Bio) using the SMARTer™ ICELL8 3’ DE Kit (Takara Bio). Processing 8 samples at a time, 80µL of each 50,000 cell/mL cell sample, negative control, positive control (K-562 RNA), and fiducial reagent (dye control) were loaded into each well of a 384-well plate (A1 through D2) and dispensed 50nL per well into a 5,184 well SMARTer™ ICELL8® 3’ DE Chip (Takara Bio) using the ICELL8® Multisample NanoDispenser (MSND, Takara Bio). After dispense, chip was sealed with imaging film, and centrifuged at 300 x g, 5 minutes, 4°C. ICELL8® 3’ DE Chip was then imaged on an ICELL8® Imaging System (Takara Bio) featuring an Olympus BX43 upright florescent microscope equipped with an automated stage, camera, DAPI, and Texas Red filters and controlled by CellSelect® Software (Takara Bio) to visually identify and select wells containing one viable single-cell candidate per well (positive Hoechst 33342, negative Propidium Iodide) generating single-cell candidate list for each ICELL8® 3’ DE Chip. After imaging, ICELL8® 3’ DE Chip were stored at -80°C overnight. Each sample was ran twice on separate ICELL8® 3’ DE Chips in order to multiplex samples and minimize chip-to-chip variations.
The ICELL8® 3’ DE Chip is preprinted with a reverse transcription (RT) primer containing a unique molecular identifier (UMI), 11-base unique well barcode, and poly T sequence in each of the 5,184 wells. The ICELL8® 3’ DE Chip containing cells was thawed at room temperature for 10 minutes, then centrifuged for 3 minutes, 3,200 x g, 4°C and maintained on ice. The ICELL8® 3’ DE Chip was placed on the ICELL8® MSND and loaded 50 µL of RT-PCR solution (Takara Bio; 56 µL 5M GC Melt, 24 µL 25mM dNTP Mix, 3µL 1M MgCl2, 9µL 100mM DTT, 62 µL 5X First-Strand Buffer, 33 µL 2X SeqAmp PCR Buffer, 16 µL 10% Triton X-100, 2 µL SMARTer ICELL8 3’ DE Oligo Mix, 29 µL 100U/µL SMARTScribe Reverse Transcriptase, 10µL SeqAMP DNA Polymerase) into four wells (A1 through A4) of a 384-well plate. MSND then dispensed 50 nL to wells containing only live single-cell candidates according to the single-cell candidate file previously generated by the ICELL8® Imaging System. The 3’ DE Chip containing single cell candidates with RT-PCR reagent sealed with PCR Sealing Film and centrifuged 3,200 x g, 3 min, 4°C and then placed in a BioRad T100 equipped with a ICELL8® Chip adapter to perform RT followed by 22 PCR cycles for cDNA amplification. Amplified cDNA from the ICELL8® 3’ DE Chip individual cells was collected and pooled using the ICELL8® Collection Kit (Takara Bio) by centrifugation at 3,200 x g, 10 minutes, 4°C. The cDNA products were concentrated using cDNA Clean & Concentrator-5 Kit (Zymo Research) followed bead cleanup using 0.6X AMPure XP Beads (Beckman Coulter) and eluted in 12 µL DNase/RNase free water. Libraries were prepared using 1ng of purified cDNA according to the ICELL8® 3’ DE instruction manual (Takara Bio) using the Nextera Primer P5 (ICELL8® 3’ DE Kit, Takara Bio), Nextera XT DNA Library Preparation Kit (Illumina) and i7 Index Primer (Nextera XT Index Kit, Illumina). Unique i7 Indexes were used for each ICELL8® 3’ DE Chip. Tagmented cDNA was amplified by PCR 12 cycles prior to dual-sided bead cleanup using AMPure XP beads, and eluted in 11µL DNase/RNase free water. Library and cDNA quality and profile were confirmed prior to sequencing ran on an Agilent Bioanalyzer using a Bioanalyzer High Sensitivity DNA Analysis kit (Agilent). Prepared library was then sequenced using 150 paired-end cycles on a NovaSeq S4 flow cell (Illumina).
single-cell RNA-Seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
single cell RNA
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| Data processing |
We demultiplexed the reads from the FASTQ files using custom code.
Samples were aligned to GRCh38 using the STAR (020201) aligner using a GTF from GENCODE
We estimated the read counts using featurecounts (v1.5.2).
We checked data quality using CollectRnaSeqMetrics and CollectAlignmentSummaryMetrics from picard (1.129), as well as with custom code.
The pipelines were created and executed using BETSY.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include read counts for each cell
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| Submission date |
Mar 21, 2020 |
| Last update date |
Jun 22, 2021 |
| Contact name |
Jeffrey Chang |
| E-mail(s) |
jeffrey.t.chang@uth.tmc.edu
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| Organization name |
University of Texas
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| Street address |
6431 Fannin St
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| City |
Houston |
| ZIP/Postal code |
77019 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE147326 |
Single Cell RNA-Seq data for Cancer Stem Like Reversal Project |
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| Relations |
| BioSample |
SAMN14420213 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4426069_Sample4.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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