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Sample GSM6940809

Query DataSets for GSM6940809

Status

Public on Mar 06, 2023

Title

10S, prefix: RIT735A4, replicate 1, scRNAseq

Sample type

SRA

Source name

trunk tissue caudal to third somite

Organism

Gallus gallus

Characteristics

cell type: trunk
tissue: trunk tissue caudal to third somite
age: chicken embryo with 10 Somites

Extracted molecule

total RNA

Extraction protocol

Fertilized chicken eggs obtained from Henry Stewart & Co. Ltd were incubated at 37°C with ~40% humidity for 29 to 45 hours to yield a minimum of 4 embryos with specific somite numbers: 4, 7, 10 and 13somites. All embryos were dissected to keep solely the tissue caudal to the third somite (inclusive). A single-cell suspension was obtained by incubating the dissected embryos at 37°C in a dissociation solution consisting of accutase (Stemcell Technologies) with 3U/mg Papain (Sigma-Aldrich, 10108014001) and 1mg/mL of Collagenase 4 (Gibco, 17104019) for 20 min. Half-way through incubation and at the end, the embryos were mechanical dissociated with a P1000 pipet. After dissociation 200uL of resuspension buffer (DMEM/F12 with 1% BSA) was added. The cell suspension was then spined for 4min at 0.6xg, resuspended in 250µL of resuspension buffer and filtered through a 40µm Flowmi cell strainer (cat. no. 136800040) and twice through a pre-wet 20µm pluriSelect strainer (43-10020-60). The yield and cell viability for the 4S, 7S, 10S and 13S samples were 640, 500, 1300 and 1600 cells/µL with a viability of 88%, 93%, 92% and 95% respectively.
A single-cell suspension was loaded independently for each sample onto the channels of Chromium Chip G for use in the 10x Chromium Controller (PN-1000120) with the goal of obtaining 10000 cells. The cells were partitioned into nanolitre scale gel beads in emulsions (GEMs) and lysed using the 10x Genomics Single Cell 3′ Chip V3.1 GEM, Library and Gel Bead Kit (PN-1000121). cDNA synthesis and library construction were performed as per the manufacturer's protocol for the Chromium Single-Cell 3′ mRNA V3.1 protocol. cDNA amplification involved 12 PCR cycles. Libraries for the samples were multiplexed so that the number of reads matched one lane per sample and sequenced on an Illumina HiSeq4000 using 100 bp paired-end runs.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

3' RNA
10x Genomics Chromium Single-Cell 3′ mRNA V3.1

Data processing

Reads were aligned to the Gallus gallus GRCg6a.101 reference genome using CellRanger (v4.0.0, 10X Genomics) and a custom-made reference 10X package including a gtf file with the protein_coding, pseudogene and lncRNA gene biotypes. Read counts were computed using DropEst61 (v0.8.6) with the parameters “-f -V -w -L eiEIBA”. The remaining analyses were performed using Scanpy62 (v1.7.0) unless otherwise indicated. Data for the different chick stages were independently filtered for high percentages of mitochondrial UMIs (6-7.5%) and low total counts (2500). Potential doublet cells were filtered out using Scrublet63 with thresholds between 0.2-0.3. Counts were normalized to a target sum of 10000 excluding 1% of highly expressed genes. “Highly variable genes” were called using Scanpy’s function with the same name with default parameters. Data of different stages were integrated using harmonypy, a port of the harmony64 R package by Ilya Korsunsky. Clustering of cells was performed unbiasedly using the Leiden algorithm with a resolution parameter of 3.5. PCA and UMAP were run with default parameters, with neighbourhood graph being computed for 10 neighbours and 40 principal components.
Assembly: Gallus gallus GRCg6a.101

Submission date

Jan 18, 2023

Last update date

Mar 06, 2023

Contact name

Tiago Rito

Organization name

The Francis Crick Institute

Lab

James Briscoe

Street address

1 Midland Rd