generation sequencing library preparation kits  (Pacific Biosciences)

Journal: Scientific Reports

Article Title: The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

doi: 10.1038/s41598-024-67242-z

Figure Lengend Snippet: The GAGsi region creates gene silencing. ( A ) Scheme of the transcriptional regulation sites of Ty1 and two types of Ty1 transcripts. The data were adapted from Curcio et al. . ( B ) Identification of the sequence responsible for silencing inside the GAG gene. Illustration of each size truncated GAG gene fragment connected to the TDH3 promoter-Flag-Venus. ( C ) Immunoblot comparing the expression levels of Flag-epitope tagging Venus (F-V), a variant of yellow fluorescent protein (YFP), in each construct illustrated in ( B ). Anti-Flag epitope antibody was used in the immunoblot. PS: Ponceau S staining to detect the proteins in each lane as loading controls. *: non-specific band. ( D ) The position of the GAGsi region in the Ty1 element. YLRWTy1-3 sequence is a representative Ty1 element used in this study. ( E ) Comparison of mRNA transcription levels. The GAL1 promoter (p GAL1 ) connected to the GAGsi- TDH3 promoter (p TDH3 )-Flag- Venus (top); GAG-p TDH3 : primer positions used in RT-PCR. mRNA transcription level comparison (bottom). + Gal: galactose used in the culture medium. + Glu: glucose used in the culture medium. The results are the average ± s. d. (n = 3, independent experiments) and analyzed by unpaired t -test (two-tailed). ( F ) Immunoblot comparing the expression levels of Flag tagging Venus (F-V) either in the presence of galactose (G) or glucose (D) used in ( E ). Anti-GFP antibody was used in the immunoblot. ( G ) Time course of yeast culture exchange from YPRG medium to YPRGD medium (left scheme). YPRG: yeast extract (Y)-polypeptone (P)-raffinose (R)-galactose (G). YPRGD: YPRG with glucose ( D ) Immunoblot of Flag tagging Venus (right). G → D: After culturing in YPRG, glucose was added to the culture medium.

Article Snippet: Adaptor and index primers were added to the sheared DNA fragments using a next-generation sequencing library preparation kit (NEBNext Ultra DNA Library Prep Kit for Illumina (Brea, CA, USA), catalog no. E7370S: NEB) according to the manufacturer’s instructions.

Techniques: Sequencing, Western Blot, Expressing, FLAG-tag, Variant Assay, Construct, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Scientific Reports

Article Title: The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

doi: 10.1038/s41598-024-67242-z

Figure Lengend Snippet: The inverted repeat (IR) sequence in the GAGsi region is necessary for the silencing effect of GAGsi. (A ) The position of the IR site in GAGsi. ( B ) The probability of IR formation by GAGsi IR wild-type sequence (upper) or the stem sequence mutant (bottom). The base-color gradation from purple to red represents an increase in the probability of IR structure. ( C ) Schematic of Mung-Bean nuclease (MBN)-PCR analysis to detect the IR structure on the plasmid. ( D ) Agarose gel electrophoresis of PCR products with or without post-MBN treatment. F-V: Flag-Venus. ( E ) Immunoblot to compare the expression levels of Flag-Venus between the wild-type and IR-stem mutant. ( F ) GAGsi sequences of various sizes centered at IR sites were connected to p TDH3 -Flag-Venus (left). Immunoblot comparing the expression levels of FLAG-tagged Venus (F-V) in each construct (right). tGAGsi: truncated GAGsi region. Anti-GFP antibody was used in the immunoblots ( E and F ).

Article Snippet: Adaptor and index primers were added to the sheared DNA fragments using a next-generation sequencing library preparation kit (NEBNext Ultra DNA Library Prep Kit for Illumina (Brea, CA, USA), catalog no. E7370S: NEB) according to the manufacturer’s instructions.

Techniques: Sequencing, Mutagenesis, Plasmid Preparation, Agarose Gel Electrophoresis, Western Blot, Expressing, Construct

Journal: Scientific Reports

Article Title: The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

doi: 10.1038/s41598-024-67242-z

Figure Lengend Snippet: The IR in GAGsi functions as an insulator. ( A ) Bisulfite-sequencing (BS-seq) analysis of GAGsi sequence. The red and yellow sequences correspond to the IR region sequences. G: G on the loop region of IR (top strand). ☆: C on loop region of IR (bottom strand). The numbers (1, 2 and 3) on the plain sequence of bottom strand indicate the sequence position on the IR structure. The number on each sequence position at the IR structure (bottom strand) indicates the CT conversion rates (%). ( B ) ChIP-qPCR analysis using anti-H3K36me3. The positions of two primer sets used in this study (upper scheme). The results are the average ± sd (n ≤ 5, independent experiments) and analyzed by unpaired t -test (one-tailed). ( C ) Comparison of the transformation (TF) efficiency between WT IR-GAGSi and mt IR-GAGsi. The stem sequence mutant was used as mt IR-GAGsi. The positions of two HIS3 gene integration sites used in this study, and the formula for calculating the TF efficiency (%) (upper scheme). The results are the average ± sd (n ≥ 5, independent experiments) and analyzed by unpaired t -test (one-tailed). The raw data is listed in Table S3C. ( D ) Schematic of IR sequence as an insulator. (E ) The IR sequence functions as an insulator. An additional GAGsi IR sequence (either wild-type or stem-mutant) was inserted between the GAGsi and TDH3 promoters as the 2nd IR (left cartoon). Immunoblot using an anti-GFP antibody to detect the expression of Flag-Venus (right).

Article Snippet: Adaptor and index primers were added to the sheared DNA fragments using a next-generation sequencing library preparation kit (NEBNext Ultra DNA Library Prep Kit for Illumina (Brea, CA, USA), catalog no. E7370S: NEB) according to the manufacturer’s instructions.

Techniques: Methylation Sequencing, Sequencing, One-tailed Test, Comparison, Transformation Assay, Mutagenesis, Western Blot, Expressing

Journal: Scientific Reports

Article Title: The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator

doi: 10.1038/s41598-024-67242-z

Figure Lengend Snippet: GAGsi assists Ty1 transcription from the 5’-LTR. ( A ) The region from the 5’-LTR to the 3' terminus of the GAG gene in YLRWTy1-3 was connected to p TDH3 -Flag-Venus. The primers between GAGsi and p TDH3 were used for RT-PCR. (B ) RT-PCR analysis for Ty1 mRNA levels in the WT IR-GAGSi and the mt IR-GAGsi. ( C ) RT-PCR analysis for Ty1 mRNA levels of the WT IR-GAGsi in GAGsi with or without HU treatment. ( D ) RT-PCR analysis for Ty1 mRNA levels either in the WT IR-GAGSi and the mt IR-GAGsi with or without HU. The Ty1 mRNA data of the WT IR sequence and the mt IR without the HU (-HU) are the same as the Ty1 mRNA data in the WT and in the mt in ( B ). The results were average ± s. d. (n = 3, independent experiments) and analyzed by unpaired t -test (two-tailed). (E ) Immunoblot comparing the expression levels of Flag-Venus (F-V) in either WT-IR or IR-mt in 5’-LTR + GAG with or without HU treatment. Anti-GFP antibody was used in the immunoblot. ( F ) The chromatin structure surrounding GAGsi region.

Article Snippet: Adaptor and index primers were added to the sheared DNA fragments using a next-generation sequencing library preparation kit (NEBNext Ultra DNA Library Prep Kit for Illumina (Brea, CA, USA), catalog no. E7370S: NEB) according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Two Tailed Test, Western Blot, Expressing