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Sample GSM4476667

Query DataSets for GSM4476667

Status

Public on Jun 30, 2021

Title

mt_d12_B2

Sample type

SRA

Source name

IVC embryo

Organism

Macaca fascicularis

Characteristics

developmental stage: Day 12
genotype: ISL1_hypomorphic_mutant
batch: B2

Treatment protocol

To generate ISL1 hypomorphic mutants, the zygotes were injected with mix of SpCas9 mRNA (200 ng/μL) and gRNAs (25 ng/μL each). Intracytoplasmic injections were performed by using the Nikon microinjection system under standard conditions. The embryos were cultured in embryo culture medium-9 (HECM-9) containing 10% fetal calf serum (Hyclone Laboratories) in 37°C incubator supplying 5% CO2. Genetic modified embryos with high quality from morula to blastocyst stage were used for further studies.

Growth protocol

Frozen NHP blastocysts at 5-7 days post fertilization were thawed by Thawing Media (Kizatato, VT802) and cultured in an optimized condition based on a human embryo culture protocol. Briefly, blastocysts were cultured in blastocyst culture medium (BCM) for at least 4 hours to recover. Then they were treated with Acidic Tyrode’s solution (Sigma Aldrich, T1788) to remove the Zona pellucida, and transfer to a ibiTreat 8-well μ-plate (Ibidi) with 300 µL of pre-equilibrated in vitro culture medium 1 (IVC1). On the second day, carefully remove 150 µL of IVC1 and supply with 200 µL pre-equilibrated IVC2. Check the blastocyst growth and change medium every two days until the end of the experiments. IVC1: sterile filtered Advanced DMEM/F12 (Thermo Fisher Scientific), 20% (vol/vol) heat-inactivated FBS (Corning), 2 mM L-Glutamine (Thermo Fisher Scientific), 1x Penicillin/Streptomycin (Thermo Fisher Scientific), 1X ITS-X (Thermos Fisher Scientific). Add 8 nM β-estradiol (Sigma Aldrich), 200 ng/ml Progesterone (Sigma Aldrich), and 25 μM N-aceyl-L-cysteine (Sigma Aldrich) to the pre-filtered medium. IVC2: sterile filter Advanced DMEM/F12 (Thermo Fisher Scientific), 30% (vol/vol) KnockOut Serum Replacement (Thermo Fisher Scientific), 2 mM l- Glutamine (Thermo Fisher Scientific), 1x Penicillin/Streptomycin (Thermo Fisher Scientific), 1X ITS-X (Thermo Fisher Scientific). Add 8nM β-estradiol (Sigma Aldrich), 200 ng/ml Progesterone (Sigma Aldrich), and 25 μM N-aceyl-L-cysteine (Sigma Aldrich) to the pre-filtered medium.

Extracted molecule

polyA RNA

Extraction protocol

Blastocysts at Day 10, Day 12 and Day 14 were washed with PBS and treated with TrypLE Express Enzyme (Thermo Fisher Scientific) for 30 minutes at 37℃. Then dissociate single cells by mouth pipetting until no bulky cell clumps could be observed. Single cells were transferred into a RNase-free, low-cell-adhesion 1.5 mL tube and centrifuged at 300 ℊ for 5 minutes. Carefully remove the supernatant and resuspended the cell pellet with 40 µL of 2% BSA in PBS. Within 30 minutes after dissociation, cells were loaded into the 10x Genomics Chromium system.
Libraries for sequencing were prepared using the Chromium Single Cell 3ʹ Reagent Kits v3 according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Sequencing data was aligned and quantified by using the Cell Ranger Pipeline v3.1.0 (10x Genomics) with standard parameters.
Ambient RNA contamination was estimated through the levels of choriogonadotropins expression in epiblast (POU5F1 positive) cells and removed from the count matrix using SoupX (https://github.com/constantAmateur/SoupX).
Filtering of the count matrix was carried out as follows: Genes were kept if they showed expression in at least 3 cells per batch. Cells were kept when they had a mitochondiral gene count percentage of < 7.5% and > 2500 expressed genes (WT_d10_b1, WT_d10_b2, WT_d12_b1, mt_d10_b1) or > 1500 expressed genes (WT_d12_b2, WT_d14_b1, WT_d14_b2, mt_d12_b1, mt_d12_b2, mt_d14_b2) or > 1000 expressed genes (mt_d14_b1)
Genome_build: Macaca_fascicularis_5.0 release 96
Supplementary_files_format_and_content: CM_filtered_uncoreccted.tsv.gz file contains a UMI (unique molecular identifier) read counts matrix of all samples after filtering genes and cells as described above.
Supplementary_files_format_and_content: CM_filtered_SoupX_corrected.tsv.gz file contains a UMI (unique molecular identifier) read counts matrix of all samples after correction for ambient RNA and filtering genes and cells as described above.

Submission date

Apr 14, 2020

Last update date

Jun 30, 2021

Contact name

Alexander Goedel

E-mail(s)

alexander.goedel@ki.se

Organization name

Karolinska Institutet

Department

Department of Cell and Molecular Biology (CMB)