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| Status |
Public on Oct 01, 2019 |
| Title |
BS-seq worker pupa (experiment 1) |
| Sample type |
SRA |
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| Source name |
Worker pupa
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| Organism |
Apis mellifera |
| Characteristics |
tissue: pupa
developmental stage: pupa
caste: worker
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| Treatment protocol |
NA
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| Growth protocol |
European honey bees were grown in Langstroth hives at the Harry H. Laidlaw Jr. Honey Bee Research Facility at University of California Davis.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worker pupae were collected at about pink eyes developmental stage. All samples were flash frozen in liquid nitrogen immediately upon collection. Except for queen heads, for which we used a single head per experiment, the rest of the biological sampling consisted of a mix of individuals, three larvae/pupae/heads, 50 embryos, and 20ul of semen. About 500 ng of genomic DNA was isolated from various honey bee developmental stages, fragmented by sonication, end repaired, and ligated to custom synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (QIAGEN), as outlined in the manufacturer’s instructions. The bisulfite-converted libraries were then amplified by PCR using ExTaq DNA polymerase (Takara Bio) for 12–14 cycles. The enriched libraries were purified using the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). About 500 ng of genomic DNA was isolated from various honey bee developmental stages, fragmented by sonication, end repaired, and ligated to custom synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (QIAGEN), as outlined in the manufacturer’s instructions. The bisulfite-converted libraries were then amplified by PCR using ExTaq DNA polymerase (Takara Bio) for 12–14 cycles. The enriched libraries were purified using the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent).
Sequencing on the Illumina GAII and HiSeq 2000 platform was performed at the Vincent J. Coates Genomic Sequencing Laboratory at the University of California, Berkeley, USA (UC Berkeley) to generate single-end 50, 76 and 100 base reads.
Honey bee samples were collected in two rounds (for two biological replicates). Worker embryos (eggs) were transferred from honeycomb cells into microcentrifuge tubes (50 per/tube). Drone and worker larvae were obtained at about 3rd and 4th instar developmental stages. Worker pupae were collected at about pink eyes developmental stage. Worker heads were collected from bees newly emerged from their own growing cells. Drone heads were collected from the same colony as drone larvae and worker embryos, larvae, pupae, and adult heads were collected from. Semen was collected from mature drones essentially as described in (Robinson et al., 2000). All samples were flash frozen in liquid nitrogen immediately upon collection. Except for queen heads, for which we used a single head per experiment, the rest of the biological sampling consisted of a mix of individuals, three larvae/pupae/heads, 50 embryos, and 20ul of semen.
BS-Seq: About 500 ng of genomic DNA was isolated from various honey bee developmental stages, fragmented by sonication, end repaired, and ligated to custom synthesized methylated adapters (Eurofins MWG Operon) according to the manufacturer’s (Illumina) instructions for gDNA library construction. Adaptor-ligated libraries were subjected to two successive treatments of sodium bisulfite conversion using the EpiTect Bisulfite kit (QIAGEN), as outlined in the manufacturer’s instructions. The bisulfite-converted libraries were then amplified by PCR using ExTaq DNA polymerase (Takara Bio) for 12–14 cycles. The enriched libraries were purified using the solid-phase reversible immobilization method using AM-Pure beads (Beckman Coulter) prior to quantification with a Bioanalyzer (Agilent). Sequencing on the Illumina GAII and HiSeq platform was performed at the Vincent J. Coates Genomic Sequencing Laboratory at the University of California, Berkeley, USA (UC Berkeley) to generate single-end 76 and 100 base reads.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
bs_exp1_wpu
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| Data processing |
Bisulfite-Seq: We used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted reference scaffold (Amel_4.5), allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases, 100 base reads were divided in half and 50 base reads were processed as single reads. Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first.
Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T.
Identifying differential methylation between various samples was performed by Fisher Exact Test using the number of Cs (methylated cytosines) and Ts (unmethylated cytosines) of any pair of samples. A site was determined to be differentially methylated if its p-value was lower than 0.001 in both biological replicates.
Percent methylation change: This number was calculated by dividing the difference in methylation level between two samples by the level of methylation in the sample with the higher methylation level. For example, percent-methylation-change between embryo and W. larva was calculated as follows: Embryo mCG-W.larva mCGEmbryo mCG ifEmbryo mCG>W.larva mCG -W.larva mCG-Embryo mCGW.larva mCGifEmbryo mCG<W.larva mCG
Gene expression: Raw Illumina RNA-Seq 100 base reads were first mapped to the most recent honey bee genome assembly (Amel_4.5; (Elsik et al., 2014)) using Tophat (Trapnell et al., 2012), with the following changes to the default Tophat v2.0.1 parameters –I 100000 (maximum intron length) and --no-novel-juncs (limiting the alignment to v3.2 honey gene annotation). Gene expression abundance and changes were calculated using the Cufflinks and Cuffdiff softwares (Trapnell et al., 2012) based on the honeybee 4.5 genome, as well as the v3.2 gene annotation file and default parameters with the addition of --min-reps-for-js-test. Differential expression was considered as statistically significant when the q-value (FDR correction) was lower than 0.05 and the FPKM fold change between two samples was higher than two.
Genome_build: Amel_4.5
Genome_annotation: OGS 3.2 scaffolds
Supplementary_files_format_and_content: GFF - Annotation of methylated CG sites
Supplementary_files_format_and_content: TXT (tab-delimited table) - Table of FPKM and differential expression of genes (rows) for each or between pairs of samples (cols).
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| Submission date |
Jul 05, 2018 |
| Last update date |
Oct 02, 2019 |
| Contact name |
Assaf Zemach |
| E-mail(s) |
assafze@tauex.tau.ac.il
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| Organization name |
Tel Aviv University
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| Department |
School of Plant Sciences and Food Security
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| Street address |
Haim Levanon
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| City |
Tel Aviv |
| ZIP/Postal code |
69978 |
| Country |
Israel |
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| Platform ID |
GPL13725 |
| Series (1) |
| GSE116629 |
DNA methylation is maintained with high fidelity in the honey bee germline and exhibits global non-functional fluctuations during somatic development |
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| Relations |
| BioSample |
SAMN09603757 |
| SRA |
SRX4341976 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3244417_wpu_exp1_methyl-group.all.single-c-CG.gff.gz |
120.5 Mb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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