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Status |
Public on Nov 04, 2024 |
Title |
10x_RGC_live_AC |
Sample type |
SRA |
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Source name |
retina
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Organism |
Mus musculus |
Characteristics |
tissue: retina gender: Male age: Postnatal month 4 genotype: wild-type treatment: CD90.2 enrichment
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Extracted molecule |
polyA RNA |
Extraction protocol |
We have generated 4 scRNA-seq samples of the mouse C57BL6/J retina. After dissection, retinas were dissociated into single cells using papain-based enzyme following the published protocol 10. With activated 45U of papain (Worthington, Cat. #LS003126) solution (1mg L-Cystine, Sigma; 8 KU of DNase I, Affymetrix; in 5 ml DPBS), retina was incubated at 37C for ~20min. Which was followed by the replacement of incubation buffer with 2ml ovomucoid solution (15 mg ovomucoid, Worthington Biochemical; 15 mg BSA Thermo Fisher Scientific; in 10 ml DPBS) and 500ul deactivated FBS. The retina tissue was carefully triturated and filtered by 20 um plastic mesh. Repeat the step with additional 1ml ovomucoid solution until no tissue is visible. Spin the cells down at 300g, 4C for 10 min, and cells were used in the next step. To deplete the photoreceptors, cells were resuspended in 0.5% BSA and stained with CD73-PE antibody for 10min at 4C (for each million cells, add 98ul 0.5% BSA with 2ul CD73-PE antibody), and washed with 35 ml 0.5% BSA at 4C for 10min. After being stained with Anti-PE microbeads (for each million cells, add 80ul 0.5% BSA and 20ul microbeads), for 15 min at 4C, cells were washed and resuspended in 0.5% BSA. CD73 negative neuronal cells were enriched by autoMACS Pro Separator (Miltenyi Biotec) DEPLETES mode and ready for the next step. Similarly, CD90.2 positive neuronal cells were enriched with CD90.2 microbeads (for each 10million cells, 90ul 0.5% BSA and 10ul CD90.2 microbeads were used) and autoMACS POSSEL-S mode. Cells viability was 85%-87% when checked with DAPI staining under microscope. Nanopore library was generated from scRNA-seq cDNA following the Nanopore Single-cell transcriptomics with cDNA prepared using 10x Genomics protocol (version Jan2022) using the SQK-PCS111 Ligation Sequencing Kit. 35-55 fmol of the library was sequenced on PromethION FLO-PRO002 R9.4.1 flow cells. The following options were used: 72hr of run time, and real-time basecalling with High-accuracy mode. Nanopore Protocol for Single-cell transcriptomics with cDNA prepared using 10X Genomics
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
PromethION |
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Description |
10x Genomics retina_isoform.gtf retina_isoform_quantify_cellclass
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Data processing |
Raw sequencing reads are processed by Sicelore pipeline (https://github.com/ucagenomix/sicelore) Transcript isoform identification and quantification using Flair (https://flair.readthedocs.io/en/latest/modules.html) Isoform QC and filtering using SQANTI3 (https://github.com/ConesaLab/SQANTI3) Assembly: mm10 (https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-mm10-2020-A.tar.gz) Supplementary files format and content: A .gtf file of information for isoforms and a cell class by isoform count matrix
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Submission date |
Feb 09, 2024 |
Last update date |
Nov 04, 2024 |
Contact name |
Rui Chen |
E-mail(s) |
ruic20@hs.uci.edu
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Phone |
7137985194
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Organization name |
Baylor College of Medicine
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Department |
Department of Molecular and Human Genetics
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Lab |
Dr. Rui Chen's lab
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Street address |
One Baylor Plaza
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City |
Houston |
State/province |
Texas |
ZIP/Postal code |
77030 |
Country |
USA |
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Platform ID |
GPL26624 |
Series (1) |
GSE255520 |
Comprehensive characterization of transcript isoform in mouse retina with ONT long read single-cell RNA sequencing |
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Relations |
BioSample |
SAMN39909222 |
SRA |
SRX23592464 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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