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| Status |
Public on Jul 23, 2020 |
| Title |
HCT-116_SP1-HyPBase_scRNA |
| Sample type |
SRA |
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| Source name |
HCT-116
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT-116
tissue: colorectal cancer
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| Treatment protocol |
HCT-116 cells were transfected using Neon electroporation with the aforementioned settings. Each replicate for electroporation was comprised of 2e6 cells. For single cell calling cards analysis of SP1 binding in HCT-116 cells, we transfected four replicates each with 5 µg PB-SRT-Puro and 5 µg SP1-HyPBase. These libraries were not mixed. We used the demultiplexed data from the cell line mixing experiment with HyPBase as controls. Cells were cultured independently and mixed immediately prior to generating single cell emulsions. All cells were allowed to recover for 24 hours before undergoing puromycin selection. A negative control replicate, transfected only with PB-SRT-Puro, was treated identically in parallel. Replicates were harvested once the negative control cells had died.
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| Growth protocol |
Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% of either penicillin-streptomycin or antibiotic-antimycotic. Cells were grown at 37ºC with 5% carbon dioxide (CO2). Media was replenished every 2 days.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell RNA-seq libraries were prepared using 10x Genomics’ Chromium Single Cell 3’ Library and Gel Bead Kit. Each replicate was targeted for recovery of 6,000 cells. Library preparation followed a modified version of the manufacturer’s protocol. We prepared the Single Cell Master Mix without RT Primer, replacing it with an equivalent volume of Low TE Buffer. Gel-in-emulsion (GEM) generation and GEM-RT incubation proceeded as instructed. At the end of Post GEM-RT cleanup, we added 36.5 µl Elution Solution I and transferred 36 µl of the eluted sample to a new tube (instead of 35.5 µl and 35 µl, respectively). The eluate was split into two 18 µl aliquots and kept at –20ºC until ready for further processing. One fraction was kept for single cell calling cards library preparation, while the other half was further processed into a single cell RNA-seq library.
We added the RT Primer sequence to the products of the scRNA-seq aliquot. We created an RT master mix by adding 20 µl of Maxima 5X RT Buffer, 20 µl of 20% w/v Ficoll PM-400, 10 µl of 10 mM dNTPs, 2.5 µl RNase Inhibitor and 2.5 µl of 100 µM 10x_TSO. To this solution we added 18 µl of the first RT product and 22 µl of ddH2O. Finally, we added 5 µl Maxima H Minus Reverse Transcriptase, mixed by flicking, and centrifuged briefly. This reaction was incubated at 25ºC for 30 minutes followed by 50ºC for 90 minutes and heat inactivated at 85ºC for 5 minutes. The solution was purified using DynaBeads MyOne Silane following 10x Genomics’ instructions, beginning at “Post GEM-RT Cleanup – Silane DynaBeads” step D. The remainder of the single cell RNA-seq protocol, including purification, amplification, fragmentation, and final library amplification, followed manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Single cell RNA-seq library
SRRs represent biological replicates.
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| Data processing |
scRNA-seq libraries were sequenced on either Illumina HiSeq 2500 or NovaSeq S1 machines. Reads were analyzed using 10x Genomics’ Cell Ranger with the following settings: --expect-cells=6000 --chemistry=SC3Pv2 --localcores=16 --localmem=30. The digital gene expression matrices from 10x were then further processed with scanpy (Wolf et al., 2018) for identification of highly variable genes, batch correction, dimensionality reduction, and Louvain clustering. Processed scRNA-seq datasets were stored as .loom files (http://loompy.org). We cross-referenced gene expression data with published datasets (Rosenberg et al., 2018; Rouillard et al., 2016; Saunders et al., 2018; Tasic et al., 2018; Zeisel et al., 2018) to assign cell types.
Genome_build: hg38
Supplementary_files_format_and_content: Loom files contain processed scRNA-seq data
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| Submission date |
Apr 10, 2020 |
| Last update date |
Jul 23, 2020 |
| Contact name |
Robi D Mitra |
| E-mail(s) |
rmitra@wustl.edu
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| Organization name |
Washington University in St. Louis
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| Department |
Genetics
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| Street address |
4515 McKinley Ave
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| City |
St. Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63143 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE148448 |
Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells |
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| Relations |
| BioSample |
SAMN14573314 |
| SRA |
SRX8094412 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4471647_HCT-116_SP1-HyPBase_scRNA.loom.gz |
1.3 Gb |
(ftp)(http) |
LOOM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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