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| Status |
Public on Jul 08, 2019 |
| Title |
latTI2_rep2 |
| Sample type |
SRA |
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| Source name |
late tail bud I_whole embryo
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| Organism |
Ciona intestinalis |
| Characteristics |
developmental stage: late tail bud I
tissue: whole embryo
replicates: biological replicate 2
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For each sample, 100 to 500 morphologically normal embryos were transferred into tubes pre-coated with 5% BSA in Ca2+-free artificial sea water (Ca2+-free ASW, 10mM KCl, 40mM MgCl2, 15mM MgSO4, 435 mM NaCl, 2.5mM NaHCO3, 7mM Tris base, 13mM Tris-HCl). Embryos were immediately dissociated with 0.5 to 1% trypsin in Ca2+-free ASW with 5 mM EGTA (ASW-EGTA) for 2min (gastrula and neurula stages) to 10min (tailbud stages). Embryos were pipetted 5min on ice to complete dissociation of individual cells. Then, the digestion was inhibited with 0.2% BSA in Ca2+-free ASW or quenched by 20% FBS. Cells were collected by centrifugation at 4°C at 500g for 2 to 5min and then resuspended in ice-cold Ca2+-free ASW containing 0.5% BSA. For the swimming larva stage, the embryos were either homogenized (H100, Waverly) and dissociated using 1% trypsin or dissociated with 1% trypsin, 1mg/ml collagenase, 0.5% pronase and 0.5mg/ml cellulase in ASW EGTA. Once dissociated, the enzymes were inhibited by 20% FBS and 2mg/ml Glycine.
For all the samples, cells were lysed, cDNAs were barcoded and amplified with Chromium Single Cell 3’ Library and Gel Bead Kit v2 (10x Genomics, CA) following the instructions of the manufacturer. Illumina sequencing libraries were prepared from the cDNA samples using the Nextera DNA library prep kit (Illumina, CA). All the libraries were sequenced on Illumina HiSeq 2500 Rapid flowcells (Illumina Inc., CA) with paired-end 26nt + 125nt reads following standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
latTI2_S1_L001
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| Data processing |
Basecalls performed using RTA version 1.18.64.0
Concatenated fastq files from both lanes of flowcell
Demultiplexed reads using barcode_splitter 0.18.2 (https://bitbucket.org/princeton_genomics/barcode_splitter)
Single cell analysis (alignment, cell identification, gene counts, etc.) performed by CellRanger count version 2.0.1
Single cell analysis by Seurat (v2.3.4) and URD (v1.0.0)
Genome_build: Genome used was (Ciona intestinalis (KH2012, Ghost Database) plus mchsv40 and cfpsv40 inserts
Supplementary_files_format_and_content: 10x Cell Ranger HDF5 formatted matrix files
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| Submission date |
May 13, 2019 |
| Last update date |
Jul 10, 2019 |
| Contact name |
Chen Cao |
| E-mail(s) |
chencao@princeton.edu
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| Organization name |
Princeton University
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| Department |
Lewis-Sigler Institute for Integrative Genomics
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| Lab |
Levine Lab
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| Street address |
Icahn lab
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| City |
Princeton |
| State/province |
New Jersey |
| ZIP/Postal code |
08550 |
| Country |
USA |
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| Platform ID |
GPL23102 |
| Series (1) |
| GSE131155 |
Comprehensive single cell transcriptome lineages of a proto-vertebrate |
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| Relations |
| BioSample |
SAMN11637724 |
| SRA |
SRX5827318 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3764780_latTI2_raw_gene_bc_matrices_h5.h5 |
28.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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