Fetal retinas (FD59, FD82 and FD125) were dissected and dissociated using Trypsin for 15–20 minutes. Cell pellets were resuspended in PBS containing 0.04% bovine serum albumin and filtered through a 35μm cell strainer (Fisher, 08–771-23) to remove cell clumps. Organoids or retinospheres were processed similarly.;10–20 organoids or retinospheres were randomly chosen (to account for any variability within the organoids or spheres) and digested using the papain dissociation kit (Worthington, #LK003150).Cells were strained through the cell strainer and counted with a hemocytometer. Typically, 2000–4000 cells of the sample were then input into the 10X protocol. GEM generation, reverse transcription, cDNA amplification, and library construction steps were performed according to manufacturer’s instructions (Chromium Single Cell 3’ v1/v2/v3 platform (10X Genomics, Pleasanton, CA). Sequencing libraries were generated using Illumina 75 cycle high sequencing kit (#20024906) and samples were run on an Illumina NextSeq 500.
Chromium single cell 3 v1 v2 v3 platform
Manufactured by 10x Genomics
The Chromium Single Cell 3' v1/v2/v3 platform is a lab equipment product offered by 10x Genomics. It is designed to enable single-cell RNA sequencing analysis. The core function of this platform is to capture and analyze the transcriptome of individual cells.
Automatically generated - may contain errors
2 protocols using chromium single cell 3 v1 v2 v3 platform
1
Single-cell RNA-seq of Fetal Retinal Cells
2
Single-cell RNA-seq of Fetal Retinal Cells
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Fetal retinas (FD59, FD82 and FD125) were dissected and dissociated using Trypsin for 15–20 minutes. Cell pellets were resuspended in PBS containing 0.04% bovine serum albumin and filtered through a 35μm cell strainer (Fisher, 08–771-23) to remove cell clumps. Organoids or retinospheres were processed similarly.;10–20 organoids or retinospheres were randomly chosen (to account for any variability within the organoids or spheres) and digested using the papain dissociation kit (Worthington, #LK003150).Cells were strained through the cell strainer and counted with a hemocytometer. Typically, 2000–4000 cells of the sample were then input into the 10X protocol. GEM generation, reverse transcription, cDNA amplification, and library construction steps were performed according to manufacturer’s instructions (Chromium Single Cell 3’ v1/v2/v3 platform (10X Genomics, Pleasanton, CA). Sequencing libraries were generated using Illumina 75 cycle high sequencing kit (#20024906) and samples were run on an Illumina NextSeq 500.
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