Fflinks2 [22], specifically Cuffdiff, to generate normalized expression (FPKM) values. The samples from which these mRNA sequences are derived are described above in the section on RNA sequencing and transcript assembly (accessions GenBank:SRX092158, GenBank:SRX092159 and GenBank: SRX101672). We also obtained 60 peripheral blood mononuclear cell (PBMC) samples from rhesus macaques in a social hierarchy experiment performed at the Yerkes National Primate Research Center. We individually subjected 10 macaques that were dominant in the social hierarchy and 10 subordinate macaques to a human intruder as a stressor. Whole blood was then collected at several time points using a BD CPT vacutainer which allow for thecollection of PBMCs. RNA was purified from PBMCs using the RNeasy kit (QIAGEN, Valencia CA). We prepared libraries using standard TruSeq chemistry (Illumina Inc., San Diego CA) and sequenced them PRIMA-1 on an Illumina Hi-Seq 1000 as 2 ?100 base paired-end reads at the Yerkes NHP Genomics Core Laboratory (http:// www.yerkes.emory.edu/nhp_genomics_core/). Sequences were deposited at NCBI under accessions [GenBank: SAMN02743270 - SAMN02743329]. We mapped reads with STAR [33] (version 2.3.0e) to both the rheMac2 and MacaM genomes, using the reference annotations as splice junction references. rheMac2 annotations were obtained from UCSC. We discarded un-annotated noncanonical splice junctions, non-unique mappings and discordant paired-end mappings. We performed transcript assembly, abundance estimates and differential expression analysis with Cufflinks2 (version 2.1.1) and Cuffdiff 2 [22]. We determined differentially expressed transcripts for pair-wise experimental group comparisons with an FDR-corrected p-value (q-value) <0.05. We compared differences in unique read counts and mapping percentages between rheMac2 and MacaM assemblies using a paired T-test.Statement of ethical approvalMaterials used in these studies were from animal work performed under Institutional Animal Care and Use Committee approval from the University of Nebraska Medical Center, Oregon Health and Sciences University and Yerkes National Primate Research Center. Animal welfare was maintained by following NIH (Public Health Service, Office of Laboratory Animal Welfare) and USDA guidelines by trained veterinary staff and researchers under Association for Assessment and Accreditation of Laboratory Animal Care certification, insuring standards for housing, health care, nutrition, environmentalTable 2 Assembly statistics for de novo rhesus transcriptsSample accession SRX099247 SRX101205 SRX101272 SRX101273 SRX101274 SRX101275 SRX103458 SRX101672 SRX092157 SRX092159 SRX092158 Tissue Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Caudate nucleus Caudate nucleus Cerebral cortex Thymus Testis Read length 76 76 76 76 76 76 76 76 100 100 100 Single or paired Single Single Single Single Single Single Paired Paired Paired Paired Paired N50 1578 1552 1659 1656 2054 1646 2651 2831 2322 2731 1970 Median 547 530 496 484 590 466 824 1079 829 1060 641 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/18218841 Mean within 1 SD 643 624 626 596 722 585 952 1128 924 1121Zimin et al. Biology Direct 2014, 9:20 http://www.biologydirect.com/content/9/1/Page 7 ofTable 3 Mutations in the reference rhesus macaque which interfere with annotationChromosome 1 1 3 9 10 15 Location PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9547713 135383631 169148554 50555959 18304595 23470221 274407 Gene symbol KLHDC9 ZBTB41 ZXDC PRUNE2 C10orf67 PRPF6 Mutation stop-gain start-loss.
A Ciência & Ensino é uma publicação semestral destinada a professores de ciências do ensino fundamental e médio e seus formadores.
Fflinks2 [22], specifically Cuffdiff, to generate normalized expression (FPKM) values. The samples
por Chun Rawls (2024-05-16)
Fflinks2 [22], specifically Cuffdiff, to generate normalized expression (FPKM) values. The samples from which these mRNA sequences are derived are described above in the section on RNA sequencing and transcript assembly (accessions GenBank:SRX092158, GenBank:SRX092159 and GenBank: SRX101672). We also obtained 60 peripheral blood mononuclear cell (PBMC) samples from rhesus macaques in a social hierarchy experiment performed at the Yerkes National Primate Research Center. We individually subjected 10 macaques that were dominant in the social hierarchy and 10 subordinate macaques to a human intruder as a stressor. Whole blood was then collected at several time points using a BD CPT vacutainer which allow for thecollection of PBMCs. RNA was purified from PBMCs using the RNeasy kit (QIAGEN, Valencia CA). We prepared libraries using standard TruSeq chemistry (Illumina Inc., San Diego CA) and sequenced them PRIMA-1 on an Illumina Hi-Seq 1000 as 2 ?100 base paired-end reads at the Yerkes NHP Genomics Core Laboratory (http:// www.yerkes.emory.edu/nhp_genomics_core/). Sequences were deposited at NCBI under accessions [GenBank: SAMN02743270 - SAMN02743329]. We mapped reads with STAR [33] (version 2.3.0e) to both the rheMac2 and MacaM genomes, using the reference annotations as splice junction references. rheMac2 annotations were obtained from UCSC. We discarded un-annotated noncanonical splice junctions, non-unique mappings and discordant paired-end mappings. We performed transcript assembly, abundance estimates and differential expression analysis with Cufflinks2 (version 2.1.1) and Cuffdiff 2 [22]. We determined differentially expressed transcripts for pair-wise experimental group comparisons with an FDR-corrected p-value (q-value) <0.05. We compared differences in unique read counts and mapping percentages between rheMac2 and MacaM assemblies using a paired T-test.Statement of ethical approvalMaterials used in these studies were from animal work performed under Institutional Animal Care and Use Committee approval from the University of Nebraska Medical Center, Oregon Health and Sciences University and Yerkes National Primate Research Center. Animal welfare was maintained by following NIH (Public Health Service, Office of Laboratory Animal Welfare) and USDA guidelines by trained veterinary staff and researchers under Association for Assessment and Accreditation of Laboratory Animal Care certification, insuring standards for housing, health care, nutrition, environmentalTable 2 Assembly statistics for de novo rhesus transcriptsSample accession SRX099247 SRX101205 SRX101272 SRX101273 SRX101274 SRX101275 SRX103458 SRX101672 SRX092157 SRX092159 SRX092158 Tissue Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Cerebral cortex Caudate nucleus Caudate nucleus Cerebral cortex Thymus Testis Read length 76 76 76 76 76 76 76 76 100 100 100 Single or paired Single Single Single Single Single Single Paired Paired Paired Paired Paired N50 1578 1552 1659 1656 2054 1646 2651 2831 2322 2731 1970 Median 547 530 496 484 590 466 824 1079 829 1060 641 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/18218841 Mean within 1 SD 643 624 626 596 722 585 952 1128 924 1121Zimin et al. Biology Direct 2014, 9:20 http://www.biologydirect.com/content/9/1/Page 7 ofTable 3 Mutations in the reference rhesus macaque which interfere with annotationChromosome 1 1 3 9 10 15 Location PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/9547713 135383631 169148554 50555959 18304595 23470221 274407 Gene symbol KLHDC9 ZBTB41 ZXDC PRUNE2 C10orf67 PRPF6 Mutation stop-gain start-loss.