Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Genome assembly and annotation workflow. ( B ) An example of a DAPI-stained spread of Hi5 cell mitotic chromosomes used to determine the karyotype. ( C ) Phylogenetic tree and orthology assignment of T. ni with 18 arthropod and two mammalian genomes. Colors denote gene categories. The category 1:1:1 represents universal single-copy orthologs, allowing absence and/or duplication in one genome. N:N:N orthologs include orthologs with variable copy numbers across species, allowing absence in one genome or two genomes from different orders. Lepidoptera-specific genes are present in at least three of the four lepidopteran genomes; Hymenoptera-specific genes are present in at least one wasp or bee genome and at least one ant genome. Coleoptera-specific genes are present in both coleopteran genomes; Diptera-specific genes present in at least one fly genome and one mosquito genome. Insect indicates other insect-specific genes. Mammal-specific genes are present in both mammalian genomes. The phylogenetic tree is based on the alignment of 1:1:1 orthologs.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Staining

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: Thirty images showing the numbers of chromosomes ( N ) in Hi5 cells. N ranged from 103 to 122; mean ± S.D.=111.7 ± 5.45. Since lepidopteran cell lines are typically tetraploid, the haploid genome likely contains 28 (mean ± S.D.=27.9 ± 1.36) pairs of chromosomes.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques:

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Genomic coverage comparison of Z-linked, W-linked and autosomal contigs. Contig coverage was shuffled 1,000,000 times to calculate the coverage ratio. Outliers are not shown. ( B ) Autosomal, Z-linked and W-linked transcript abundance in Hi5 cells and T. ni tissues. ( C ) Transcript abundance ratios of autosomal, Z-linked, and W-linked genes in Hi5 cells and T. ni tissues. Error bars represent 95% confidence interval estimated from 1000 bootstrap replicates. ( D ). Sex-specific splicing of T. ni doublesex pre-mRNA.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Comparison

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Comparison of miRNA abundance in male and female T. ni thoraces. Solid circles, miRNAs with FDR < 0.1 and fold change >2. Outlined circles, all other miRNAs. ( B ) Comparison of the tissue distribution of the 44 most abundant miRNAs among T. ni ovaries, testes, and Hi5. ( C ) Heat map showing the abundance of miRNAs in ( B ). miRNAs are ordered according to abundance in ovary. Conservation status uses the same color scheme in ( A ).

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Comparison

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Abundance of mRNAs encoding piRNA pathway proteins) in Hi5 cells, ovary, testis, and thorax. ( B ) Ideogram displaying the positions of miRNA genes (arrowheads) and piRNA clusters in the T. ni genome. Color-coding reports tissue expression for Hi5 cells, ovaries, testis, and thorax. Contigs that cannot be placed onto chromosome-length scaffolds are arbitrarily concatenated and are marked ‘Un.’ ( C ) Distribution of piRNAs among the autosomes, Z, and W chromosomes in Hi5 cells, ovary, testis, and female and male thorax, compared with the fraction of the genome corresponding to autosomes, W, and Z chromosomes.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Expressing

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) piRNA clusters tend to produce piRNAs that are antisense to transposons. The x -axis represents the ratio of piRNAs from the plus strand to piRNAs from the minus strand, with the dotted lines indicating twofold difference. The y -axis indicates the ratio of transposons lengths on the plus strand over transposon length on the minus strand. The solid line indicates regression line and shading indicates 95% confidence interval by LOWESS. Boxplot shows fractions of antisense transposons (i.e. transposons inserted opposite to the direction of piRNAs precursor transcription) in dual- and uni-strand clusters. Outliers are not shown. Wilcoxon rank-sum test. ( B ) piRNA densities on autosomal, Z-linked and W-linked contigs in Hi5 cells, ovary, testis, and female and male thorax. ( C ) Abundance of piRNAs from putative W-linked genes.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques:

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Comparison of piRNA abundance (ppm) from ovary and Hi5 piRNA-producing loci and from ovary and testis piRNA-producing loci. ( B ) piRNA cluster lengths in T. ni ovary, testis, thorax, and Hi5 cells. ( C ) Motifs around intron boundaries of predicted protein-coding gene models within and outside of piRNA clusters.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Comparison

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Hi5-specific piRNA clusters contain younger transposon copies. RC, rolling-circle transposons; LINE, Long interspersed nuclear elements; LTR, long terminal repeat retrotransposon; DNA, DNA transposon. ( B ) Comparison of piRNA abundance per cluster in female and male thorax. ( C ) piRNA precursors are rarely spliced. The number of introns supported by exon-exon junction-mapping reads is shown for protein-coding genes and for piRNA clusters for each tissue or cell type. ( D ) piRNA precursors are inefficiently spliced. Splicing efficiency is defined as the ratio of spliced over unspliced reads. Splice sites were categorized into those inside and outside piRNA clusters. Outliers are not shown.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Comparison

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Strategy for using Cas9/sgRNA RNPs to generate a loss-of-function TnPiwi deletion allele. Red, protospacer-adjacent motif (PAM); blue, protospacer sequence. Arrows indicate the diagnostic forward and reverse primers used in PCR to detect genomic deletions (Δ). Sanger sequencing of the ~1700 bp PCR products validated the TnPiwi deletions. ( B ) An example of PCR analysis of a TnPiwi deletion event. ( C ) Strategy for using Cas9/sgRNA RNPs and a single-stranded DNA homology donor to insert EGFP and an HA-tag in-frame with the vasa open-reading frame. ( D ) An example of PCR analysis of a successful HDR event. DNA isolated from wild type (WT) and FACS-sorted, EGFP-expressing Hi5 cells (HDR) were used as templates.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Sequencing, Diagnostic Assay, Isolation, Expressing

Journal: eLife

Article Title: The genome of the Hi5 germ cell line from Trichoplusia ni , an agricultural pest and novel model for small RNA biology

doi: 10.7554/eLife.31628

Figure Lengend Snippet: ( A ) Schematic of single-clone selection of genome-edited Hi5 cells using the strategy described in . ( B ) A representative field of Hi5 cells edited to express EGFP-HA-Vasa from the endogenous locus. ( C ) A representative image of a fixed, EGFP-HA-Vasa-expressing Hi5 cell stained with DAPI, anti-EGFP and anti-HA antibodies. EGFP and HA staining colocalize in a perinuclear structure consistent with Vasa localizing to nuage.

Article Snippet: Forty eight hours after transfection, Hi5 cells from one 90% confluent well of a six-well plate (Corning, Corning, NY) were collected, washed once with PBS (ThermoFisher) and lysed in 2 × PK buffer containing 200 μg/ml proteinase K, extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then genomic DNA collected by ethanol precipitation.

Techniques: Selection, Expressing, Staining