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| Status |
Public on Mar 27, 2024 |
| Title |
E34_VZ_SG, rep1 |
| Sample type |
SRA |
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| Source name |
cerebral cortex
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| Organism |
Mustela putorius |
| Characteristics |
tissue: cerebral cortex
litter: 2
region: splenial gyrus
germinal zone: ventricular zone
developmental stage: embryonic day 34
sequencing flowcell: BCDM6YANXX
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After microdissecting individual germinal zones from living cortical slices, tissue pieces were enzymatically dissociated for 20-30’ at 37ºC using MACS Neural Tissue Dissociation Kit P (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were mechanically suspended using wide-tipped, fire-polished Pasteur pipettes (Fisher Scientific) followed by P1000 pipettes (Gilson). Cell suspensions were filtered to 2ml DNA LoBind tubes (Eppendorf) through 40 μm PluriStrainer Mini (PluriSelect) pre-wet with Hank’s Balanced Salt Solution (1X) without CaCl2/MgCl2/pH indicator (HBSS, Gibco). Cells were centrifuged (Spectrafuge 24D, Labnet) twice for 5min at 1000 rpm, and cell pellets were resuspended in 40μl of Phosphate Buffered Saline ph7.4 (1X) without CaCl2/MgCl2 (PBS, Gibco) containing 0.04% Bovine Serum Albumin (BSA, Sigma-Aldrich). Cell concentration from homogeneous suspensions was measured, and cell death was estimated twice using trypan blue 0.4% stain (Thermo Fisher Scientific): first on an automatic cell counter (Countess II FL, Thermo Fisher Scientific) and second using a hemocytometer (BLAUBRAND Neubauer, BRAND) on an inverted microscope (Leica DM IL). Debris-free suspensions with cell viability over 90% were used immediately for single-cell isolation.
Cell suspensions from 400 to 1200 cells/μl were loaded into the Chromium Single Cell Controller (10x Genomics) to target a recovery of 7k to 10k cells/sample. Single-cell Gel bead-in-EMulsions (GEMs) were generated, cells were lysed, and the released RNAs were retrotranscribed (RT) and barcoded inside the GEMs according to Single Cell 3’ Reagent Kits v2 and v3 (10x Genomics) protocols. Following GEMs break, barcoded cDNAs were pooled and cleaned from leftover RT reagents using DynaBeads MyOne Silane Beads (Invitrogen) and amplified (8 to 14 cDNA amplification cycles) following the Single Cell 3’ Reagent Kits v2 and v3 (10x Genomics) protocols. Enzymatic fragmentation, size selection and cleanup with SPRIselect Reagent kit (Beckman Coulter) were followed by library construction (total sample index cycles from 12 to 16 cycles), consistent with standard Illumina sequencing constructs and performed in line with Single Cell 3’ Reagent Kits v2 and v3 (10x Genomics) protocols. cDNA and library concentration and quality check were assessed using Bioanalyzer High Sensitivity DNA Kit (Agilent) and 2100 Expert Software (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
10x Genomics Single Cell 3’ Reagent Kit v2
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| Data processing |
Genome alignment: Ferret gene annotation from Johnson et al., 2018, comprising Ensembl Mustela putorius furo annotation expanded using bulk RNAseq data, was further enriched with NCBI’s RefSeq mitochondrial genes from Mustela putorius furo annotation release 101 (Accession No. NC_020638.1), and transformed from a general feature format (GFF3) into gene transfer format (GTF) using Cufflinks-2.2.1. Johnson’s GenBank accessions were matched to RefSeq accessions through cthreepo-0.1.1 and gene_name and transcript_name attibutes were added to GTF rows where they were missing by appending gene_id and transcript_id entries. scRNA-seq reads were mapped to NCBI ferret reference genome (MusPutFur1.0/GCF_000215625.1) and Unique Molecular Identifiers (UMIs) were collapsed by STAR-2.7.3a adapting the parameters to v2 or v3 10x chemistry, depending on the sample.
Cell barcode filtering: Count matrices from each sample were loaded and cell-containing droplets were identified and distinguished from empty droplets retaining ambient RNA by DropletsUtils-1.6.1. Increased niters argument to 100,000 and decreased false discovery rate (FDR) to 0.1% were applied to all samples. A lower bound of 80 was implemented for replicates P1_VZ_LS_rep1 and E34_VZ_SG_rep1. Mitochondrial genes were removed before cell calling for replicates E34_VZ_LS_rep2, P1_VZ_SG_rep2, P1_VZ_LS_rep3, P1_OSVZ_SG_rep1 and P1_OSVZ_LS_rep1 to promote potential unhealthy cell removal. The automatic retention of droplets with large total UMI counts (determined by the knee point) was disabled for replicate P1_VZ_LS_rep3.
Assembly: MusPutFur1.0/GCF_000215625.1
Supplementary files format and content: Tab-separated values files from cell barcodes and genes (barcodes.tsv, genes.tsv) and matrix file (matrix.mtx) for each sample after genome alignment and cell barcode filtering.
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| Submission date |
Dec 02, 2022 |
| Last update date |
Mar 27, 2024 |
| Contact name |
Lucia Del-Valle-Anton |
| Organization name |
Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández
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| Lab |
Borrell Lab
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| Street address |
Av. Santiago Ramón y Cajal s/n
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| City |
Sant Joan d’Alacant |
| ZIP/Postal code |
03550 |
| Country |
Spain |
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| Platform ID |
GPL32908 |
| Series (2) |
| GSE219254 |
scRNA-seq of ferret cortical germinal zones from splenial gyrus and lateral sulcus (Borrell lab) |
| GSE234305 |
Multiple parallel cell lineages in the developing mammalian cerebral cortex |
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| Relations |
| BioSample |
SAMN32007789 |
| SRA |
SRX18473127 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6779217_E34_VZ_SG_1_barcodes.tsv.gz |
7.2 Kb |
(ftp)(http) |
TSV |
| GSM6779217_E34_VZ_SG_1_genes.tsv.gz |
208.6 Kb |
(ftp)(http) |
TSV |
| GSM6779217_E34_VZ_SG_1_matrix.mtx.gz |
9.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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