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| Status |
Public on Jul 23, 2020 |
| Title |
Mouse-Cortex_HyPBase_scRNA |
| Sample type |
SRA |
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| Source name |
Mouse cortex
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| Organism |
Mus musculus |
| Characteristics |
tissue: cerebral cortex
strain: C57BL6/J
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| Treatment protocol |
We mixed equal volumes of each virus and performed intracranial cortical injections of the mixture into newborn wild-type C57BL/6J pups (P0-2). As a gating control, we injected one litter-matched animal with AAV9-PB-SRT-tdTomato only. After 2 to 4 weeks, we sacrificed mice and dissected the cortex (8 libraries) or hippocampus (1 library).
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| Growth protocol |
All mouse experiments were done following procedures described in (Cammack et al., 2019). In brief, we cloned the PB-SRT-tdTomato and HyPBase constructs into AAV vectors. The Hope Center Viral Vectors Core at Washington University in St. Louis packaged each construct in AAV9 capsids. Titers for each virus ranged between 1.1e13 and 2.2e13 viral genomes/ml. All animal practices and procedures were approved by the Washington University in St. Louis Institutional Animal Care and Use Committee (IACUC) in accordance with National Institutes of Health (NIH) guidelines.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mouse cortical tissues were dissociated to single suspensions following a modification of previously published methods (Avey et al., 2018; Saxena et al., 2012). We incubated samples in a papain solution containing Hibernate-A with 5% v/v trehalose, 1x B-27 Supplement, 0.7 mM EDTA, 70 µM 2-mercaptoethanol, and 2.8 mg/ml papain. After incubation at 37ºC, cells were treated with DNaseI, triturated through increasingly narrow fire-polished pipettes, and passed through a 40-micron filter prewetted with resuspension solution: Hibernate-A containing 5% v/v trehalose, 0.5% Ovomucoid Trypsin Inhibitor, 0.5% Bovine Serum Albumin (BSA), 33 µg/ml DNaseI (Worthington), and 1x B-27 Supplement. The filter was washed with 6 ml of resuspension solution. The resulting suspension was centrifuged for 4 minutes at 250 g. The supernatant was discarded. The pellet was then resuspended in 2 ml of resuspension solution and resuspended by gentle pipetting. We eliminated subcellular debris using gradient centrifugation. We first prepared a working solution of 30% w/v OptiPrep Density Gradient Medium mixed with an equal volume of 1x Hank’s Balanced Salt Solution (HBSS) with 0.5% BSA. We then prepared solutions of densities 1.057, 1.043, 1.036, and 1.029 g/ml using by combining the working solution with resuspension solution at ratios of 0.33:0.67, 0.23:0.77, 0.18:0.82, and 0.13:0.87, respectively. We layered 1 ml aliquots of each solution in a 15 ml conical tube beginning with the densest solution on the bottom. The cell suspension was added last to the tube and centrifuged for 20 minutes at 800g at 12ºC. The top layer was then aspirated and purified cells were isolated from the remaining layers. These cells were then resuspended in FACS buffer: 1x HBSS, 2 mM MgCl2, 2 mM MgSO4, 1.25 mM CaCl2, 1 mM D-glucose, 0.02% BSA, and 5% v/v trehalose. Cells were centrifuged for 4 minutes at 250 g, the supernatant was discarded, and the pellet was resuspended in FACS buffer by gentle pipetting. Cells were then sorted based on fluorescence activity. As a gating control, we analyzed cells from cortices injected with AAV9-PB-SRT-tdTomato only. We then collected cells from brains transfected with AAV9-PB-SRT-tdTomato and AAV9-HyPBase whose fluorescence values exceeded the gate. After sorting, cells were centrifuged for 3 minutes at 250 g. The supernatant was discarded and cells were resuspended in FACS buffer at a concentration appropriate for 10x Chromium 3’ scRNA-seq library preparation. 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 NovaSeq 6000 |
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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: mm10
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 |
GPL24247 |
| 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 |
SAMN14573321 |
| SRA |
SRX8094424 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4471659_Mouse-Cortex_HyPBase_scRNA.loom.gz |
276.6 Mb |
(ftp)(http) |
LOOM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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