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| Status |
Public on Feb 24, 2021 |
| Title |
invivo_crispr_ko_trim28fl_823 |
| Sample type |
SRA |
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| Source name |
invivo_crispr_ko_trim28fl_823_R1
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| Organism |
Mus musculus |
| Characteristics |
experiment: In vivo CRISPR Trim28fl
condition: KO
genotype/variation: Trim28 floxed mice
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| Treatment protocol |
In vitro CRISPR: Transduced with a lentiviral vector expressing gRNAs (LV.gRNAs) designed to target either exon 3, 4 or 13 of Trim28 or to target lacZ as well as a nuclear RFP reporter gene (H2B-RFP). Lentiviruses were produced according to Zufferey et al and titers were 10^9 TU/ml, which was determined using qRT-PCR. Cas9 mouse NPCs where transduced at a MOI 40 and allowed to expand for 10 days prior to FACS. In vivo CRISPR Trim28fl: AAV vectors expressing Cre were injected into the forebrain of adult floxed Trim28 animals. The production of AAV5 vectors has been described in detail elsewhere (Ulusoy et al 2009) and titers were in the order of 1013 TU/ml, which was determined by qRT-PCR using taqman primers towards the ITR . Prior to injection, the vectors were diluted in PBS; the vectors containing the guide RNAs were diluted to 30% except upon co-injection of guides 3, 4, and 13 where the vectors were diluted to 10% each. Rosa26 Cas9 knock-in mice where anesthetized by isoflurane prior to the intra-striatal injections (coordinates from bregma: AP +0.9 mm, ML +1.8 mm, DV -2.7 mm) of 1 ml virus solution (0.1 µl/15 s). The needle was kept in place for additional 2 min post injection to avoid backflow. In vivo CRISPR Cas9: We used AAV vectors to express guides 3, 4 and 13 (targeting exon 3, 4 and 13 of Trim28) and H2B-RFP under Synapsin. The production of AAV5 vectors has been described in detail elsewhere (Ulusoy et al 2009) and titers were in the order of 10^13 TU/ml, which was determined by qRT-PCR using taqman primers towards the ITR . Prior to injection, the vectors were diluted in PBS; the vectors containing the guide RNAs were diluted to 30% except upon co-injection of guides 3, 4, and 13 where the vectors were diluted to 10% each. Rosa26 Cas9 knock-in mice where anesthetized by isoflurane prior to the intra-striatal injections (coordinates from bregma: AP +0.9 mm, ML +1.8 mm, DV -2.7 mm) of 1 ml virus solution (0.1 µl/15 s). The needle was kept in place for additional 2 min post injection to avoid backflow. In vivo CRISPR Stop Cas9: We generated AAV-vectors expressing the gRNAs and a Cre-inducible H2B-RFP reporter, as well as an AAV-vector expressing Cre under the control of the neuron-specific Synapsin (SYN) promoter. The production of AAV5 vectors has been described in detail elsewhere (Ulusoy et al 2009) and titers were in the order of 10^13 TU/ml, which was determined by qRT-PCR using taqman primers towards the ITR . Prior to injection, the vectors were diluted in PBS; the vectors containing the guide RNAs were diluted to 30% except upon co-injection of guides 3, 4, and 13 where the vectors were diluted to 10% each. Rosa26 Cas9 knock-in mice where anesthetized by isoflurane prior to the intra-striatal injections (coordinates from bregma: AP +0.9 mm, ML +1.8 mm, DV -2.7 mm) of 1 ml virus solution (0.1 µl/15 s). The needle was kept in place for additional 2 min post injection to avoid backflow. Cut and Run (IgG and H3K9me3): The Cut & Run were performed according to Skene et al 2018 . In brief, 200.,000 mouse NPCs were washed, permeabilized and attached to ConA-coated magnetic beads (Bang Laboratories) before incubated with the H3K9me3 (1:50, ab8898, Abcam) antibody at 4°C overnight. Cells were washed and incubated with pA-MNase fusion protein and digestion was activated by adding CaCl2 at 0°C. The digestion was stopped after 30 min and the target chromatin released from the insoluble nuclear chromatin before extracting the DNA. Emx animals (single cell): Animals were sacrificed by cervical dislocation and brains quickly removed. The desired regions were dissected and snap frozen on dry ice and stored at -80°C.
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| Growth protocol |
In vitro CRISPR: NPC cultures from Rosa26-Cas9 knock-in transgenic mice (embryonic day 13.5). The forebrain was mechanically dissociated and plated in gelatin coated flasks and maintained as a monolayer culture in NSA medium (Euromed, Euroclone) supplemented with N2 hormone mix, EGF (20 ng/ml; Gibco), bFGF (20 ng/ml; Gibco), 2 nM L-glutamine and 100 ug/ml Pen/Strep. Cells were then passaged 1:3-1:6 every 2-3 days using Accutase (Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro CRISPR: Cells were detached and resuspended in basic culture media (media excluding growth factors) with propidium iodide (1:1,000; BD Biosciences) and strained (70 mm filters, BD Biosciences). RFP+ cells were FACS isolated at 4°C (reanalysis showed >99% purity) and pelleted at 1,500 rpm for 5 min, snap frozen on dry ice and stored at -80°C until RNA/DNA were isolated. For all in vivo CRISPR experiments animals were sacrificed after two months and analyzed either by IHC or nuclei isolation (see details below) followed by DNA- or RNA-seq. Emx animals (RNAseq): Animals were sacrificed at 3 months of age for either immunohistochemistry IHC or RNA sequencing. Emx animals (single cell): The nuclei isolation was performed according to Sodersten et al. (2018). The tissue was thawed and dissociated in ice-cold lysis buffer ((0.32 M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM DTT) using a 1 ml tissue douncer (Wheaton). The homogenate was carefully layered on top of a sucrose cushion (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl, pH 8.0, and 1 1 mM DTT) before centrifugation at 30,000 × g for 2 hours and 15 min. Pelleted nuclei were softened for 10 minutes in 100 ml of nuclear storage buffer (15% sucrose, 10 mM Tris-_HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) before resuspended in 300 ml of dilution buffer (10 mM Tris-_HCl, pH 7.2, 70 mM KCl, and 2 mM MgCl2) and run through a cell strainer (70 mm). Cells were run through the FACS (FACS Aria, BD Biosciences) at 4°C with low flowrate using a 100 mm nozzle (reanalysis showed >99% purity).
RNAseq: Total RNA was isolated from frozen cell/nuclei pellets and brain tissue using the RNeasy Mini Kit (Qiagen) and used for RNA-seq and qPCR (tissue pieces were run in the tissue lyser for 2 min, 30 Hz, prior to RNA isolation). Single cell: 8,500 nuclei per sample were sorted via FACS and loaded onto 10X Genomics Single Cell 3’ Chip along with the reverse transcription mastermix following the manufacturer’s protocol for the Chromium Single Cell 3′ Library (10X Genomics, PN-120233) to generate single-cell gel beads in emulsion. cDNA amplification was done as per the guidelines from 10x Genomics and sequencing libraries were generated with unique sample indices (SI) for each sample.
RNAseq libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2 × 150bp). Cut and run (IgG and H3K9me3) library preparation was performed using the Hyper prep kit (KAPA biosystems) and sequenced on NextSeq500 2 x 75 bp. For the single cell sequencing, libraries for samples were multiplexed and sequenced on a Novaseq using a 150-cycle kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file:
invivo_crispr_trim28fl.cntTable.txt
gene_count_matrix_2.csv
fullMMERVK10C_count_matrix_2.csv
ERVK_count_matrix_2.csv
TE_count_matrix_2.csv
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| Data processing |
Unique mapping - RNAseq (STAR aligner (2.6.0))
Quantification of reads mapping to genes - RNAseq (featureCounts from Subread (1.6.3))
Quantification of reads mapping to TEs - RNAseq (featureCounts from Subread (1.6.3))
Quantification of reads mapping to ERVKs- RNAseq (featureCounts from Subread (1.6.3))
Quantification of reads mapping to full MMERVK10C - RNAseq (featureCounts from Subread (1.6.3))
Multi mapping - RNAseq (STAR aligner (2.6.0))
Quantification of reads mapping to genes and TE subfamilies - RNAseq (TEtranscripts from TEToolkit (2.0.3))
Base calling, demultiplexing and convertion to fastq - single cell (cellranger mkfastq (3.0))
Read quantification - single cell (cellranger count (3.0))
Unique mapping - Cut and run (Bowtie2 (2.3.4.2))
Subtraction of input signal - Cut and run (BamCompare from deeptools)
Compute mean of signals for the different guide RNAs - RNAseq (invitro CRISPR NPC)
computeMatrix for plotHeatmap of full length MMERVK10C elements (deeptools) - RNAseq (invitro CRISPR NPC) and Cut and Run
Genome_build: GRCm38.p6 vM19
Supplementary_files_format_and_content: counts matrix; binary count matrix for CR020_S9 and CR021_S10
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| Submission date |
Jul 10, 2020 |
| Last update date |
Feb 26, 2021 |
| Contact name |
Johan Jakobsson |
| E-mail(s) |
johan.jakobsson@med.lu.se
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| Organization name |
Lund University
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| Department |
Wallenberg Neuroscience Center
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| Lab |
Molecular Neurogenetics
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| Street address |
Sölvegatan 17
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| City |
Lund |
| ZIP/Postal code |
22362 |
| Country |
Sweden |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE154196 |
Activation of endogenous retroviruses during brain development causes neuroinflammation |
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| Relations |
| BioSample |
SAMN15502823 |
| SRA |
SRX8707025 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4665757_invivo_crispr_trim28fl_ko_823.bw |
16.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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