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Mouse Tmem150a Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Tmem150a Knockout Project (CRISPR/Cas9) Objective: To create a Tmem150a knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Tmem150a gene (NCBI Reference Sequence: NM_144916.3 ; Ensembl: ENSMUSG00000055912 ) is located on Mouse chromosome 6. 8 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 8 (Transcript: ENSMUST00000069695). Exon 3~5 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 3 starts from about 8.12% of the coding region. Exon 3~5 covers 24.97% of the coding region. The size of effective KO region: ~1295 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 8 Legends Exon of mouse Tmem150a Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 409 bp section upstream of Exon 3 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 291 bp section downstream of Exon 5 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(409bp) | A(24.45% 100) | C(25.67% 105) | T(20.78% 85) | G(29.1% 119) Note: The 409 bp section upstream of Exon 3 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(291bp) | A(26.46% 77) | C(26.46% 77) | T(21.99% 64) | G(25.09% 73) Note: The 291 bp section downstream of Exon 5 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 409 1 409 409 100.0% chr6 + 72356314 72356722 409 browser details YourSeq 27 31 58 409 100.0% chr13 + 50743260 50743589 330 browser details YourSeq 22 99 121 409 100.0% chr2 + 117344927 117344951 25 browser details YourSeq 22 341 362 409 100.0% chr17 + 24187386 24187407 22 browser details YourSeq 21 104 124 409 100.0% chr17 + 81471236 81471256 21 browser details YourSeq 21 10 36 409 88.9% chr16 + 16958909 16958935 27 browser details YourSeq 20 341 360 409 100.0% chr11 - 23381788 23381807 20 browser details YourSeq 20 340 359 409 100.0% chr10 - 88659718 88659737 20 Note: The 409 bp section upstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 291 1 291 291 100.0% chr6 + 72358018 72358308 291 browser details YourSeq 26 227 259 291 96.5% chr12 + 47083863 47083902 40 browser details YourSeq 25 234 270 291 74.1% chr3 + 122086600 122086629 30 browser details YourSeq 25 54 79 291 100.0% chr1 + 193147364 193147411 48 browser details YourSeq 21 117 139 291 95.7% chr7 + 42072650 42072672 23 browser details YourSeq 21 50 70 291 100.0% chr2 + 147288843 147288863 21 browser details YourSeq 20 246 267 291 95.5% chr5 - 43082005 43082026 22 browser details YourSeq 20 68 91 291 91.7% chr8 + 84953683 84953706 24 browser details YourSeq 20 256 275 291 100.0% chr8 + 77782089 77782108 20 browser details YourSeq 20 55 74 291 100.0% chr10 + 67484281 67484300 20 Note: The 291 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Tmem150a transmembrane protein 150A [ Mus musculus (house mouse) ] Gene ID: 232086, updated on 26-Jun-2020 Gene summary Official Symbol Tmem150a provided by MGI Official Full Name transmembrane protein 150A provided by MGI Primary source MGI:MGI:2385244 See related Ensembl:ENSMUSG00000055912 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Tmem150; BC014685 Expression Broad expression in placenta adult (RPKM 123.6), liver adult (RPKM 91.9) and 23 other tissues See more Orthologs human all Genomic context Location: 6; 6 C1 See Tmem150a in Genome Data Viewer Exon count: 8 Annotation release Status Assembly Chr Location 108.20200622 current GRCm38.p6 (GCF_000001635.26) 6 NC_000072.6 (72355483..72359762) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 6 NC_000072.5 (72305477..72309756) Chromosome 6 - NC_000072.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Tmem150a ENSMUSG00000055912 Description transmembrane protein 150A [Source:MGI Symbol;Acc:MGI:2385244] Gene Synonyms Tmem150 Location Chromosome 6: 72,355,447-72,359,762 forward strand. GRCm38:CM000999.2 View alleles of this gene on alternative sequences About this gene This gene has 5 transcripts (splice variants), 1 gene allele, 246 orthologues, 4 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Tmem150a- ENSMUST00000069695.8 1546 271aa ENSMUSP00000063977.2 Protein coding CCDS20239 Q91WN2 TSL:1 201 GENCODE basic APPRIS P1 Tmem150a- ENSMUST00000132243.2 1478 168aa ENSMUSP00000138445.1 Protein coding - S4R204 TSL:5 202 GENCODE basic Tmem150a- ENSMUST00000206531.1 544 174aa ENSMUSP00000145673.1 Protein coding - A0A0U1RNR4 CDS 3' 204 incomplete TSL:5 Tmem150a- ENSMUST00000206064.1 540 120aa ENSMUSP00000146268.1 Protein coding - A0A0U1RQ67 CDS 3' 203 incomplete TSL:5 Tmem150a- ENSMUST00000206821.1 551 No - Processed - - TSL:5 205 protein transcript Page 7 of 9 https://www.alphaknockout.com 24.32 kb Forward strand 72.350Mb 72.355Mb 72.360Mb 72.365Mb Genes (Comprehensive set... 0610030E20Rik-204 >retained intron Tmem150a-202 >protein coding 0610030E20Rik-203 >retained intron Tmem150a-201 >protein coding 0610030E20Rik-201 >protein coding Tmem150a-203 >protein coding 0610030E20Rik-202 >retained intron Tmem150a-205 >processed transcript Tmem150a-204 >protein coding Contigs AC116115.11 > Genes < Gm45051-201antisense < Rnf181-201protein coding < Vamp5-203protein coding (Comprehensive set... < Rnf181-202protein coding < Vamp5-202protein coding < Rnf181-214retained intron < Vamp5-201protein coding < Rnf181-204nonsense mediated decay < Rnf181-207protein coding < Rnf181-212protein coding < Rnf181-210nonsense mediated decay < Rnf181-206nonsense mediated decay < Rnf181-205nonsense mediated decay < Rnf181-203protein coding < Rnf181-211nonsense mediated decay < Rnf181-209retained intron < Rnf181-208retained intron < Rnf181-213retained intron Regulatory Build 72.350Mb 72.355Mb 72.360Mb 72.365Mb Reverse strand 24.32 kb Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding processed transcript Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000069695 4.32 kb Forward strand Tmem150a-201 >protein coding ENSMUSP00000063... Transmembrane heli... Low complexity (Seg) Pfam Frag1/DRAM/Sfk1 PANTHER PTHR21324 Transmembrane protein 150A All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 271 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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  • SUPPLEMENTAL METHODS Negative Selection of Human

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    SUPPLEMENTAL METHODS Negative selection of human primary CD4 T cells. Buffy coats were incubated with 1/10 volume of RosetteSep CD4 T cell enrichment cocktail (StemCell Technologies Inc., Vancouver, Canada) at room temperature for 20 min and diluted with one volume of phosphate buffered saline (PBS) containing 2% human AB serum (2% HAB/PBS). CD4 T cells were separated from the remainder of peripheral blood mononuclear cells (PBMCs) and red cell rosettes by Ficoll (Histopaque-1077, Sigma Chemical Company, St. Louis, MO) density gradient centrifugation and washed twice with 2% HAB/PBS. Cell pellets were resuspended in RPMI 1640 medium supplemented with 2 mM glutamine, 100 U/ml penicillin/100 μg/ml streptomycin, and 5% human AB serum, and left at 37oC overnight prior to histone deacetylase inhibitor (HDACi) treatment the next day. Analysis of microarray gene expression data. The quality of microarray data was assessed by constructing MA-plots using the affyPLM package [1] and by unsupervised clustering. The Bioconductor package lumi [2] was used to transform (i.e. variance-stabilizing) and normalize (i.e. robust-spline) the data and remove genes not expressed in any of the samples. Technical batch effects were removed using ComBat [3]. Paired multivariate permutation tests implemented in BRB-Array Tools (version 4.2.1) [4] under default settings (80% confident no more than 10% false positives) were used to identify genes significantly differentially expressed between SAHA treated and untreated samples. Pathway analysis was performed using gene set expression comparison tool in BRB-Array Tools. All pre-defined Kyoto Encyclopedia of Genes and Genomes (KEGG) [5, 6] and BioCarta pathways (http://cgap.nci.nih.gov/Pathways/BioCarta_Pathways) were analyzed between SAHA-treated and untreated samples.