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Sample GSM8073709 Query DataSets for GSM8073709
Status Public on Nov 04, 2024
Title 10x_RGC_live_AC
Sample type SRA
 
Source name retina
Organism Mus musculus
Characteristics tissue: retina
gender: Male
age: Postnatal month 4
genotype: wild-type
treatment: CD90.2 enrichment
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
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model PromethION
 
Description 10x Genomics
retina_isoform.gtf
retina_isoform_quantify_cellclass
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
 
Submission date Feb 09, 2024
Last update date Nov 04, 2024
Contact name Rui Chen
E-mail(s) ruic20@hs.uci.edu
Phone 7137985194
Organization name Baylor College of Medicine
Department Department of Molecular and Human Genetics
Lab Dr. Rui Chen's lab
Street address One Baylor Plaza
City Houston
State/province Texas
ZIP/Postal code 77030
Country USA
 
Platform ID GPL26624
Series (1)
GSE255520 Comprehensive characterization of transcript isoform in mouse retina with ONT long read single-cell RNA sequencing
Relations
BioSample SAMN39909222
SRA SRX23592464

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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