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H2afb3 Gm14920 H2afb2 Pseudo M R N a E X P Re S S Io N Re La Tiv E to M

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H2afb3 Gm14920 H2afb2 Pseudo M R N a E X P Re S S Io N Re La Tiv E to M Expression level of H2A.B.3 in the testis of FVB/NJArc mice 450 400 350 300 250 200 150 100 50 mRNA expression relativetomHPRT mRNA 0 H2Afb3 gm14920 H2Afb2 Pseudo H2A.B.3 genes Supplementary Figure 1: The relative expression of H2A.B.3 gene family members in the testis of FVB/NJArc mice. Gene expression was assessed relative to mHPRT using qPCR. 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Supplementary Figure 2. TALEN pair specificity tested by the DLLS assay. TALEN pairs designed to target the H2Afb3 gene only (Group 1) or all three H2A.B.3 genes (Group 2). One day before transfection, 20,000 cells per well were seeded into a 96-well plate. Cells were transiently transfected with four plasmids including a pair of TALENs, the SSA Firefly luciferase reporter, and the internal control Renilla Luciferase reporter. The luciferase assay was performed 24 hr post-transfection. The pair 101421:101422 was used to create the H2A.B.3 KO mouse. Supplementary Figure 3. Cel 1 assays. Neuro2a cells were transiently transfected with various TALEN pairs and extracted genomic DNA was amplified by PCR with gene-specific primers and subjected to Cel1 digestion. TALEN activity is represented by the % cleaved (% genomic mutation, GM) shown below each lane. Cleaved DNA products (arrow heads), uncut DNA (arrow). TALEN pair 101421:101422, highlighted in red, showed the highest activity for all three H2A.B.3 genes. 1,2,3 and P denotes H2Afb3, gm14920, H2Afb2 and pseudo H2A.B.3 genes, respectively. Two biological replicates are shown. Empty GFP-vector was used as a negative control. 101408 CGCCGCCACCGTCGCT ! !!! 101407 CGCCGCCACCGTCG! !!!!! 101421 CCCGCACCTCCAGAG! H2Afb3 !ATGCCAAGGAACAGGGAAAACTGTCTTCGAGAGTCTTCAGGTCGCCGCCACCGTCGCTCCCGCACCTCCAGAGCT! gm14920 !---------------------------------------------------------------------------! H2Afb2 !-------------C--------------A-AG-----------A------A-A--A-----------------G-! Pseudo !----------G--CA------A-------T-G-G--------T--A------T-T------:------------C! ! ! CGATTAGAAACGACAC 101410! ! TTAGAAACGACAC 101412! H2Afb3 !GAGCTAATCTTTGCTGTGAGCCTGGTGGAACAGCATCTGAGGGAGGTTAGCCGTGCCCGGAGGCTCAGTGATACG! gm14920 !-----G--------A------------------------------A-----------T---------------T-! H2Afb2 !-----G--------A----------------------------------------------------------T-! Pseudo !-----G------A-A------------------AG---------CC------A----T---------------T-! ! CACTCGGACCACCTTG 101422! !!! ! !!!!! ! H2Afb3 !GTGCCCATCTTCCTGGCAGCCATCCTGGAGTCCCTCACCCGCAGGTTGCTGGAGCTTGCCGGCAATGAGGCCCAA! gm14920 !-----------------------------------------------------------G---------------! H2Afb2 !------G---C-----T--------------T-------A-------------------G---------------! Pseudo !-----A—T-----------------------T-------TA----C-------------G---------A----G! ! H2Afb3 !CGCAGAGGTACCGAGAGGCGCATCAACTCCTGAACTGCTGGACTTGGCTGTCTACAGCAATATGGAGCTAAGTGA! gm14920 !---------------------------------------------------------------------------! H2Afb2 !------------C------T-----------C---C-----------AG----------G---------------! Pseudo !-AG---------C---T-A----------------------------AG-----T---------C---------- ! Supplementary Figure 4. TALEN-targeted DNA sequences. The alignment of H2A.B.3 genes and the pseudogene. TALENs specific to H2afb3 are in blue and those common for all H2A.B3 genes are in red. a b NM10 NM10 NM10 L 7 6 5 L 7 6 5 L 7 6 gm14920 Fw/Rev H2afb2 Fw/Rev gm14920 Fw H2afb2 Rev c gm14920 H2afb2 Supplementary Figure 5. An example of chimeras formed between gm14920 and H2afb2 in some G1 mice produced by crossing founder L74-5 with a wt. female. (a) genes gm14920 and H2afb2 are not amplified with gene-specific primers in pups #6 and #7, respectively but is amplified in a pup #5. (b) amplification with gm14920-Fw and H2afb2-Rev primers shows the chimeric product in pups #6 and 7. (c) A schematic diagram showing that a chimera was formed between gm14920 and H2afb2 genes with a small deletion between these fused genes suggesting that NHEJ mechanisms were involved. Fw, forward primer; Rev, reverse primer; number; L, DNA ladder. G1 G2 398 419 19 306 G3 Supplementary Figure 6: Computational analysis predicted 19 putative heterozygous deletions shared between all 3 generations of H2A.B.3-/y KO mice. If TALENs introduced off-target mutations in G0 founder mice, then those mutations would be inherited by their progeny as heterozygous. detectedeach for gene by Pindel The screen shots aret mutations in H2A.B.3genes. 7 Supplementary Figure H2afb2 gm14920 H2afb3 for all H2A.B.3 genes. The orange border box indicates thedeleted region for all H2A.B.3genes. NM4:G3:32 NM4:G2:18 NM4:G1:23 NM4:G3:32 NM4:G2:18 NM4:G1:23 NM4:G3:32 NM4:G2:18 NM4:G1:23 . Exome sequencing revealsall thethree expected SAMtools . heoutputSVs call from by a H2afb3 gm14920 H2a2 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 Expression relave to HPRT 0 0 0 wt KO wt KO wt KO b wt KO 15 kDa IP: anti-H2A.B.3 10 kDa WB: anti-H2A.B.3 c ♀H2A.B.3 +/+ x ♂ H2A.B.3 +/+ ♀H2A.B.3 +/+ x ♂ H2A.B.3 -/Y Supplementary figure 8. H2A.B.3-/Y KO males have impaired fertility. (a). The mRNA level of each H2A.B.3 gene was determined by real-time quantitative PCR, relative to HPRT. Primers were designed to anneal within the TALEN induced deletions. (b). Confirmation that H2A.B.3 protein is not produced in in H2A.B.3-/Y mice. H2A.B was immunoprecipitated from total testis lysates followed by Western Blotting using anti-H2A.B.3 polyclonal antibodies. (c) The breeding was conducted using age matched wt females (3-6 months) and age matched wt (n=5) and H2A.B.3-/Y (n=5) males. Total number of pups produced was 201 and 256 for wt and KO males, ***p value=≤0.001(ANOVA test). % Gene names/length Strand Chromosome Start End Identity H2afb3, 348bp - X 117426357 117426704 100 ENSMUSG00000083616 Gm14920, 348bp + X 114128366 114128713 98.28 ENSMUSG00000067441 H2afb2, 349bp + X 113794787 113795134 92.55 EG624153 Gm14904(Pseudo), 345bp + X 113311798 113312139 87.83 ENSMUSG00000084053 Supplementary Table 1. The genomic coordinates of H2A.B.3 coding genes. The degree of homology between the genes is indicated as % Identity. Target genes Design # Targeted sequence 101406 ttCAGGTCGCCGCCACCGTcg 101407 gtCGCCGCCACCGTCGCTcc 101408 gtCGCCGCCACCGTCGCTCcc 101409 ctCACAGCAAAGATTAGCTca H2Afb3 101410 ctCACAGCAAAGATTAGCtc (Group 1) 101412 ctCACAGCAAAGATTag 101413 ctGAGCTAATCTTTGCTgt 101415 ctAATCTTTGCTGTga 101416 ctAATCTTTGCTGTGAgc 101417 ctAACCTCCCTCAGATGCTgt 101418 ctAACCTCCCTCAGATgc 101419 ctCCGGGCACGGCTAACCTcc 101421 ctCCCGCACCTCCAGAGct H2Afb3, 101422 ctGTTCCACCAGGCTCACag Gm14920, 101423 ctGCTGTTCCACCAGGCTca H2afb2 101424 ctGGTGGAACAGCATCTga (Group 2) 101426 gtATCACTGAGCCTCCgg 101427 gtTGCTGGAGCTTgc 101429 gtACCTCTGCGTTcc Supplementary Table 2. List of TALENs designed to target H2A.B.3 genes. Sample ID Total reads Read length (bp) Approximate coverage (x) NM4-G1-28 226,900,000 100 613 NM4-G2-18 228,900,000 100 619 NM4-G3-32 129,800,000 100 351 Supplementary Table 3. The exome sequencing coverage. 3 consecutive generations of mice from the NM4 H2A.B.3 KO colony were sequenced using paired-end sequencing with a 100bp read length. Sample ID SNVs Small deletions Small insertions NM4-G1-28 1,745,670 82,379 90,762 NM4-G2-18 1,768,957 81,956 90,005 NM4-G3-32 693,686 33,823 38,321 Supplementary Table 4. Putative SNVs and Indels identified in H2A.B.3 KO mice using the mm10 mouse genome as a reference. Sample ID SNVs Small deletions Small insertions NM4-G1-28 21,256 3,889 4,378 NM4-G2-18 26,646 4,064 4,445 NM4-G3-32 13,417 1,589 2,032 Supplementary Table 5. Putative SNVs and Indels identified in H2A.B.3 KO mice after applying the FVB/NJ strain filter. Genotype Homozygous Heterozygous Sample ID No Total 0/0 1/1 2/2 0/1 0/2 1/2 Call NM4-G1-28 131 732 2 388 2 27 111 1393 NM4-G2-18 127 727 3 392 3 25 116 1393 NM4-G3-32 219 527 8 306 4 15 314 1393 Supplementary Table 6. Predicted putative homozygous and heterozygous polymorphic variants (off-target deletions) in three consecutive generations of H2A.B.3-/y mice. Exome data was referenced to mm10 genome, followed by filtering of FVB/N-specific variants and analysed for genome polymorphism using Pindel tool. Homozygous polymorphisms: no polymorphic alleles (0/0), both alleles differ from reference (1/1), and both alleles differ from reference and from 1/1 (2/2). Heterozygous polymorphisms: one allele differs from the reference genome (0/1), one allele defers from the reference genome and from 0/1 (0/2), both alleles differ from the reference genome and from each other (1/2). No call, not assigned to any type. # Chromosome Locus start Locus end Deletion size (bp) Gene ID 1 1 171573961 171573965 4 ENSMUSG00000004709 2 1 173270636 173270648 12 ENSMUSG00000049605 3 1 173760152 173760189 38 ENSMUSG00000073489 4 12 115158545 115158549 5 ENSMUSG00000093894 5 12 115335195 115335199 5 ENSMUSG00000095197 6 12 115808522 115808526 21 ENSMUSG00000091087 7 12 115833994 115834007 14 ENSMUSG00000096020 8 17 23882468 23882474 7 ENSMUSG00000096445 9 17 35266213 35266218 6 ENSMUSG00000073411 10 17 36189406 36189415 10 ENSMUSG00000054128 11 17 48145527 48145531 5 ENSMUSG00000073386 12 2 119618224 119618250 27 ENSMUSG00000072980 13 3 7604075 7604079 5 ENSMUSG00000040329 14 4 41195246 41195333 88 ENSMUSG00000028433 15 4 42871823 42871856 34 ENSMUSG00000050141 16 5 113819659 113819689 31 ENSMUSG00000048163 17 7 8245023 8245027 5 ENSMUSG00000053720 18 7 47981089 47981093 5 ENSMUSG00000067173 19 X 95940663 95940678 16 ENSMUSG00000057421 Supplementary
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