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| Status |
Public on Sep 22, 2021 |
| Title |
P28_V1_Dlxpos_RNA |
| Sample type |
SRA |
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| Source name |
Cortex-V1
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| Organism |
Mus musculus |
| Characteristics |
cell type: cortical interneurons
age: P28
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For embryonic timepoints, male hemizygous Dlx6a-Cre mice (Jax stock #008199) were crossed with female homozygous Ai9 or Ai14 mice (Rosa-LSL-tdtomato, Jax stock # 007909 or 007914) and checked daily for the presence of a vaginal plug, with the morning a plug was found being considered embryonic day 0. Embryos were screened using a fluorescent microscope for the presence of tdTomato. Embryos were harvested from pregnant dams and relevant brain regions were dissected in ice-cold Leibovitz's L-15 medium. Pooled tissue from several embryos were dissociated using the Neural Tissue Dissociation Kit (T) (Miltyeni Biotec, PN-130-093-231) according to the manufacturer’s instructions. Dissociated cells were then filtered through a 70 um filter, centrifuged at 300xg for 5 min at 4C, and, where cell-sorting was required, resuspended in 1% BSA in PBS for cell sorting of tdTomato+ cells. Sorting was performed on a Sony SH800S cell sorter with a 100 µm chip. For postnatal timepoints, male hemizygous Dlx6a-Cre mice were crossed with female homozygous INTACT mice. Nuclei for scRNA-seq were isolated as described previously with some modifications. Briefly, brains were harvested, sectioned coronally on a stainless steel mouse brain slicer (Zivic Instruments), and regions of interest were dissected in ice cold Homogenization Buffer (HB) (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH, 1mM DTT, 0.15mM spermine, 0.5mM spermidine). Dissected tissue was then transferred to a 2 ml dounce homogenizer containing HB + 0.3% IGEPAL-CA630 + 0.2U/µl RNAse inhibitor and homogenized with 10 strokes of pestle A and 10 strokes of pestle B. Nuclei were then filtered through a 30 μm filter and centrifuged at 500xg for 5 minutes at 4C. The pellet was resuspended in 1% BSA + 0.2U/µl RNAse inhibitor in PBS and centrifuged again at 500xg for 5 minutes at 4C. The pellet was then again resuspended in 1% BSA + 0.2U/µl RNAse inhibitor in PBS an filtered through a 40 μm filter for sorting of GFP+ nuclei on a Sony SH800S cell sorter with a 100 µm chip. Nuclei were collected in 1% BSA + 0.2U/µl RNAse inhibitor for input to 10X Genomics scRNA-seq. (flox-Sun1-eGFP, Jax stock #021039) to yield Dlx6a-Cre;INTACT offspring.
scRNA-seq libraries were prepared using the Chromium Single Cell 3' Solution v3 (10X Genomics). Libraries were sequenced using a Nova-Seq S2 100 cycle kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw scRNA-seq data was processed using the standard CellRanger v3.0.0 pipeline (10x Genomics). FASTQ files were generated from BCL files using cellranger mkfastq.
Read filtering, alignment, and barcode counting were performed using cellranger count. For single-nuclei RNA-seq datasets, a custom pre-mRNA reference from the mm10 genome was generated allowing intronic sequences to be included in gene counts.
Genome_build: mm10
Supplementary_files_format_and_content: barcodes.tsv files are tsv files containing cell barcodes present in the corresponding expression matrix.
Supplementary_files_format_and_content: features.tsv files are tsv files containing gene IDs and names present in the corresponding expression matrix.
Supplementary_files_format_and_content: matrix.mtx files are sparse matrix files containing feature-barcode matrices.
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| Submission date |
Jan 11, 2021 |
| Last update date |
Sep 22, 2021 |
| Contact name |
Kathryn Allaway |
| Organization name |
Broad Institute
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| Department |
Stanley Center for Psychiatric Disease
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| Lab |
Fishell Lab
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| Street address |
75 Ames St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE164570 |
Single-cell RNA-seq of developing cortical interneurons |
| GSE165233 |
Genetic and epigenetic coordination of cortical interneuron development |
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| Relations |
| BioSample |
SAMN17283753 |
| SRA |
SRX9821840 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5014308_P28_V1_Dlxpos_RNA_barcodes.tsv.gz |
43.0 Kb |
(ftp)(http) |
TSV |
| GSM5014308_P28_V1_Dlxpos_RNA_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM5014308_P28_V1_Dlxpos_RNA_matrix.mtx.gz |
96.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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