|
| Status |
Public on Dec 05, 2023 |
| Title |
AAVlib2_E15_sort |
| Sample type |
SRA |
| |
|
| Source name |
Forebrain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Forebrain
genotype: C57BL/6
age: E15.5
treatment: 14 serotype AAV library injected into lateral ventricle at E13.5, transduced cells
|
| Treatment protocol |
AAV was administered in utero to the lateral ventricle at E13.5-14.5 in Cas9 transgenic mice (Jax#026179)
|
| Growth protocol |
All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of The Scripps Research Institute
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003150) in a modification of a previously described protocol. Briefly, young mice were anesthetized then disinfected with 70% ethanol and decapitated. The brains were quickly extracted and gently dabbed with a PBS-soaked Kimwipe (Kimberly-Clark) to remove the meninge and fibroblasts. Cortices were micro-dissected in ice-cold dissection medium (Hibernate A medium (Thermo Fisher Scientific, #A1247501) with B27 supplement (Thermo Fisher Scientific, #17504044) and Trehalose (Sigma Aldrich, Cat# T9531) under a dissecting microscope. Microdissected cortices were transferred into papain solution with DNase in a cell culture dish and cut into small pieces with a razor blade. The dish was then placed onto a digital rocker in a cell culture incubator for 30 mins with rocking speed at 30 rpm at 37°C. The digested tissues were collected into a 15 mL tube and triturated with a 10 mL low bind plastic pipette 20 times and the cell suspension was carefully transferred to a new 15 mL tube. 2.7 mL of EBSS, 3 mL of reconstituted Worthington inhibitor solution, and DNase solution were added to the 15 mL tube and mixed gently. Cells were pelleted by centrifugation at 300 g for 5 mins at 4°C, followed by washing with 8ml cold dissection medium at 200g for 5 min at 4°C. Cells were resuspended in 0.5 mL ice-cold dissection medium with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, #16000069) and SYTOX dead cell stain (Invitrogen, #S34859), and subjected to FACS purification using Sony cell sorter (SH800).
scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3' Solution v3.1 kit with Feature Barcode Technology or the Chromium Next GEM Single Cell 5’ Solution v2 kit with Feature Barcode Technology (10x Genomics) following the manufacturer’s protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
AAV_all.Robj
AAV_ctxobj.Robj
|
| Data processing |
BCL files of transcriptome libraries were used to generate FASTQ files using the default parameters by “cellranger mkfastq” command (Cell Ranger v7.1.0). The gRNA library or AAV barcode dial-out library was demultiplexed using bcl2fastq.
The “cellranger count” command was used to align the transcriptome reads to the mouse genome reference mm10 (GENCODE vM23/Ensembl 98) and generate a gene expression count matrix, using expect-cells = 9,000. The AAV barcodes or gRNA reads were quantified at the single-cell level with the feature-ref flag in Cell Ranger.
Assembly: mm10
Supplementary files format and content: Robj or QS file of gene-cell matrix
|
| |
|
| Submission date |
Dec 05, 2023 |
| Last update date |
Mar 25, 2024 |
| Contact name |
Xinhe Zheng |
| E-mail(s) |
xzheng@scripps.edu
|
| Organization name |
The Scripps Research Institute
|
| Department |
Neuroscience
|
| Lab |
Xin Jin
|
| Street address |
3528 General Atomics Court
|
| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92121 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE249416 |
Massively parallel in vivo Perturb-seq reveals cell type-specific transcriptional networks in cortical development |
|
| Relations |
| BioSample |
SAMN38680100 |
| SRA |
SRX22798674 |
