Additional file 2: of MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection

Total (A) and Normalized (B) Sequence Counts per Known Bovine miRNA Homolog Group. (XLSX 73 kb

Additional file 2: of What’s in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual

Note 1: The Onchocerca ochengi reference assembly is contaminated with bovine genomic sequence. Note 2: Estimation of the number of protein coding genes missing or misassembled in the UMD3.1 bovine reference assembly. (DOCX 41 kb

Additional file 1: of What’s in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual

Table S1. Statistics from the de novo assembly of unmapped reads from DNA sequencing. Table S2. Statistics from the de novo assembly of unmapped reads from RNA sequencing. Table S3. Summary of all significant alignments from pairwise alignment of de novo assembled contigs from DNA unmapped reads to the nt database. Table S4. Number of significant alignments per tissue from pairwise alignment of de novo assembled contigs from unmapped RNA-seq reads to the nt database. Table S5. Summary of all significant alignments from pairwise alignment of de novo assembled contigs from unmapped RNA-seq reads to the nt database. Table S6. DNA sequencing metadata. Table S7. Genes represented in alignments of de novo assembled contigs from unmapped RNA-seq reads to Bos taurus. Table S8. Genes represented in alignments of de novo assembled contigs from unmapped RNA-seq reads to Bison bison bison, Bubalus bubalis, or Bos mutus. (XLSX 731 kb

Additional file 1: Table S1. of Reduced representation bisulphite sequencing of ten bovine somatic tissues reveals DNA methylation patterns and their impacts on gene expression

Tissue samples used in the RRBS analysis. Table S2. RRBS validation results. Table S3. Different methylated cytosine information. Table S4. Significantly correlated DMCs and gene information. Table S5. Different methylated CpG island information. Table S6. Significantly correlated DMIs and gene information. Table S7. Tissue-specific different methylated cytosine information. Table S8. Tissue-specific different methylated CpG island information. Table S9. Primers used for validation of RRBS results. (XLS 2962 kb

Additional file 2: of Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep

Table S2. Summary of predicted of off-targets and de novo variants. (XLSX 587 kb

Additional file 1: of Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep

Supporting figures and tables. Figure S1. Sanger sequencing confirms the SNVs identified by WGS in three trios. Example of SNVs sequenced in trio members. Figure S2. Sanger sequencing validation of the genetic modification SNVs in the offspring of #28 (#171001, #171004, and #171018). Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicate deletions, and lowercases indicate insertions or replacements. Deletions (−) and mutations (m) are shown to the right of each allele. The genotypes are shown to the right with the rates of total clones for TA-sequencing. Figure S3. Sanger sequencing validation of the genotypes of six SNVs in the offspring of #28 (#171001, #171004, and #171018). Figure S4. Sanger sequencing confirms the edited sites (indels) in each gene identified by WGS in three trios. The upper windows were generated by using Integrative Genomics Viewer (IGV) browser ( http://software.broadinstitute.org/software/igv/ ). Target sequences complementary to sgRNAs of targeted genes are in red text, while the PAM sequences are marked in green. The mutations are marked in blue, dashlines indicate deletions, and lowercases indicate insertions or replacements. Figure S5. Genome-wide distribution of putative off-target sites in the three trios used for WGS. Putative off-target sites were identified by aligning the sgRNA sequences to the sheep reference genome (Oar v3.1) allowing for a maximum of five mismatches. Potential off-target sites predicted by both Cas-OT and Cas-OFFinder are displayed using the OmiCircos tool ( http://bioconductor.org/packages/release/bioc/html/OmicCircos.html ). De novo indels in the founder animals #25 (A), #28 (B), and #A9 (C). Putative off-target sites in ASIP (a), BCO2 (b), and MSTN (c). The position of three targeted genes was highlighted with black dots. Figure S6. Validation of the 2.4 kb inversion in 54 animals. The founder animal #25 was marked with red color. Table S1 Detailed information of the three trios for whole-genome sequencing. (DOCX 1633 kb

mRNA expression levels of NRCAM, PNPLA8, CTTNBP2.

<p>Graph created using data from <a href="http://www.biogps.org" target="_blank">www.biogps.org</a> (accessed 7/18/2012) for mRNA expression levels of the gene’s mRNA expression across 84 mouse and humans tissues. While few gene expression across multiple tissues have been done in bovine, it is logical to expect a similar expression pattern as shown above given the high gene exon homology of PNPLA8, CTTNBP2, and NRCAM between bovine, mouse, human, and other mammalian species (ENSEMBL).</p

Additional file 1 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing

Additional file 1: Fig S1. Editing efficiency in the generated lambs as shown by targeted deep sequencing. Fig. S2. Detection of potential off-target sites by Sanger sequencing in founder animals. Five potential off-target sites (Spacer OT1-OT5) were predicted by Cas-OFFinder in FecBB-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the five founder animals. Fig. S3. Detection of potential off-targeted sites by Sanger sequencing in founder animals. Nine potential off-target sites (Nick OT1-OT9) were predicted by Cas-OFFinder in FecBB-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the five founder animals. Fig. S4. Detection of potential off-targeted sites by Sanger sequencing in founder animals. A potential off-target site (Spacer OT1) was predicted by Cas-OFFinder in TBXT-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the three founder animals. Fig. S5. Detection of potential off-targeted sites by Sanger sequencing in founder animals. Fifteen potential off-target sites (Nick1 OT1-OT8 and Nick2 OT1-OT7) were predicted by Cas-OFFinder in TBXT-edited lambs. Sanger sequencing was used to determine variations at predicted target sites for the three founder animals. Fig. S6. Digital PCR of PE edited cell populations (Sp1-1, Sp1-2, and Sp1-3) and an unedited wild type control. (a, d, g, j, and m) chip view by calls. (b, c, e, f, h, I, k, l, n, and o) yellow: no amplification, red: VIC reporter dye signal (KCNJ5 WT probe), blue: FAM reporter dye signal (KCNJ5 G151R probe), green: FAM + VIC reporter dye signals. (b) Two-dimensional scatter plot of digital PCR SNP assay and (c) histogram from unedited wild type control cells. (e) Two-dimensional scatter plot of digital PCR SNP assay and (f) histogram from replicate Sp1-1. (h) Two-dimensional scatter plot of digital PCR SNP assay and (i) histogram from replicate Sp1-1 (repetition of dPCR chip). (k) Two-dimensional scatter plot of digital PCR SNP assay and (l) histogram from replicate Sp1-2. (n) Two-dimensional scatter plot of digital PCR SNP assay and (o) histogram from replicate Sp1-3. Fig. S7. Sanger sequencing of all ten porcine blastocysts shows a heterozygous p.G151R mutation next to the silent mutation of the DdeI restriction enzymatic digestion site. Fig. S8. Sanger sequencing of colonies after cloning of blastocyst PCR products into the pGEM-T Easy vector. (a) WT1b_3A_a3 and (b) WT1b_3A_a5 show overlapping sequences besides sequences containing both desired mutations and sequences matching to the wild-type reference sequence

Additional file 2 of Generation of sheep with defined FecBB and TBXT mutations and porcine blastocysts with KCNJ5G151R/+ mutation using prime editing

Additional file 2: Table S1. List of primers used for genotyping and amplifying PE3-targeted FecBB fragment in HEK293T cells. Table S2. Sequences of pegRNA and sgRNA used in human HEK293T cells. Table S3. Sequences of pegRNAs and sgRNAs used in sheep. Table S4. List of primers used for in vitro transcription. Table S5. List of primers for genotyping and amplifying PE3-targeted BMPR1B and TBXT fragments in newborn lambs. Table S6. List of predicted off-target sites for PE3-targeted BMPR1B. Table S7. List of predicted off-target sites for PE3-targeted TBXT. Table S8. List of primers for genotyping and amplifying predicted off-target site fragments in FecBB-edited sheep. Table S9. List of primers for genotyping and amplifying predicted off-target site fragments in TBXT-edited sheep. Table S10. Sequences of pegRNAs and sgRNAs used in pigs. Table S11. List of primers for genotyping and amplifying PE3-targeted KCNJ5 in porcine kidney fibroblasts and blastocysts. Table S12. Number of blastocysts generated per SCNT session using KCNJ5G151R/+ porcine kidney fibroblasts as donor cells