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| Status |
Public on May 23, 2022 |
| Title |
E. coli, K12 strain, clone 1, bisulfited, ABBS primer |
| Sample type |
SRA |
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| Source name |
Bacterial cells
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| Organism |
Escherichia coli |
| Characteristics |
treatment: Bisulfite
primer used: ABBS
library prep kit used: NEB
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| Treatment protocol |
No cell treatment was applied.
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| Growth protocol |
Escherichia coli K12 cells were acquired from Neb (#E4104), MA and two clones were obtained by streaking an E. coli suspension on a LB agar plate, and further cultured in liquid LB containing no antibiotics, at 37C. Optical density was measured at 600nm every twenty minutes until bacteria reached a plateau phase, then the culture was quickly placed on ice and spun on a Sorvall centrifuge at 7500 RPM / 4C for 10 minutes, then washed twice with ice cold 1x PBS and aliquots were snap frozen in liquid nitrogen and kept at -80C for further genomic DNA extraction. K562 cells were grown in RPMI-1640 supplemented with L-glutamine, 10% FBS and 1X penicillin G + streptomycin, at 37C / 5% CO2. Cells were then trypsinized and counted with a haemocytometer while viability was assessed with Trypan Blue staining. The culture was quickly placed on ice and spun on a Sorvall centrifuge at 3000 RPM / 4C for 5 minutes, then washed twice with ice cold 1x PBS and aliquots were snap frozen in liquid nitrogen and kept at -80C for further genomic DNA extraction.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer’s protocol.
3μg of E. coli or 10μg of K562 genomic DNA spiked with 1% of unmethylated Lambda DNA was bisulfite-converted with Zymo Lightning kit (Zymo Research, CA) following the manufacturer’s instructions, then eluted in 2x10μl of Tris-HCl 10mM pH8.0 (of note, desulfonation was performed for 20mins, on column). An additional 15-minute sonication was achieved using PIXUL Multisample sonicator (Active Motif, CA) with the default settings. DNA was heated at 95C for 3 minutes then placed on ice and quantification was done using Nanodrop (Thermo Fisher Scientific, MA) while profiles were measured with RNA TapeStation (Agilent, CA) to ensure for proper sonication. Bacterial non-converted DNA was sonicated for 30 minutes using PIXUL Multisample sonicator with the default settings and DNA was further heated at 95C for 3 minutes and stored at -80C. Second strand synthesis was performed as follow: 100ng of genomic DNA was diluted in Neb 1x NEBuffer 2, supplemented with 0.25mM dNTPs and 6.5μM (K562 cells) or 0.65μM (E. coli K12 cells) ABBS primers (NNNNNSuperG) or WGBS primers (NNNNNN) in 29 μl, then heated at 94C for 2 minutes and rapidly placed on ice for 3-5 minutes. One microliter of Neb Klenow exo minus (5 units/μl) was added to the reaction that was gradually heated from 4C to 37C at a rate of 0.1C/second. Samples were then incubated for 30 minutes at 37C and purified with 45μl of Ampure XP beads (Beckman Coulter, CA). DNA was eluted in 20μl Tris-HCl 10mM pH8.0 supplemented with 0.05% Tween-20 and quantified with Qubit 2.0 dsDNA HS kit (Thermo Fisher Scientific, MA). Double stranded library preparation was performed either with Neb NEBNext® UltraTM II DNA Library Prep Kit for Illumina or Active Motif Next Gen DNA Library Kit, following manufacturers’ recommendations, with the exception that libraries amplification was performed using KAPA HiFi HotStart Real-Time Library Amp Kit (Roche, Switzerland). After a final purification using 0.75 volumes of Ampure XP beads, libraries were resuspended in 12μl of Tris-HCl 10mM pH8.0 supplemented with 0.05% Tween-20 and quantified with Qubit 2.0 dsDNA HS kit while profiles were measured with HS dsDNA TapeStation to calculate molarity
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Raw reads (FastQ files) were aligned to human hg19 or E. coli K12 genomes using Bismark (https://github.com/FelixKrueger/Bismark) following the command line: $ bismark <genome path> -p 4 --parallel 6 -q --pbat -o <output folder> -1 read1.fastq.gz -2 read2.fastq.gz.
PCR duplicates were removed following these arguments: $ deduplicate_bismark -p --output_dir <output folder> -- bam <input bam file>
MAPQ filter of 20 was applied using samtools (https://github.com/samtools/samtools): $ samtools view -h -q 20 <input bam file> -o <output folder bam file>.
Methylated cytosines were extracted with Bismark Methylation Extractor following the following arguments: $ bismark_methylation_extractor --parallel 10 -p --no_overlap --ignore 7 -- ignore_r2 7 --ignore_3prime 5 --ignore_3prime_r2 5 -o <output folder> --report --bedGraph -- zero_based --cutoff 1 --CX --remove_spaces --cytosine_report --genome_folder <genome path> <input bam file>
bam files were sorted and indexed using samtools sort and samtools index
Bigwig coverage files were obtained with deeptools bamcoverage (https://github.com/deeptools/deepTools): $ bamCoverage -b <input bam file> -o <output folder bigwig file> --normalizeUsing RPKM --binSize 5 --numberOfProcessors 16
Genome_build: E. coli K12 or hg19
Supplementary_files_format_and_content: Bigwig (bw) files are RPKM-normalized
Supplementary_files_format_and_content: Methylation bedgraph files contain methylation percentages and coverage information
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| Submission date |
Jul 25, 2021 |
| Last update date |
May 26, 2022 |
| Contact name |
Benjamin Delatte |
| E-mail(s) |
benche65@gmail.com
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| Organization name |
Active Motif
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| Department |
Advanced Research
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| Street address |
1914 Palomar Oaks Way STE 150
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| City |
Carlsbad |
| State/province |
CA |
| ZIP/Postal code |
92008 |
| Country |
USA |
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| Platform ID |
GPL21222 |
| Series (1) |
| GSE180796 |
Anchor-Based Bisulfite Sequencing determines genome-wide DNA methylation |
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| Relations |
| BioSample |
SAMN20391565 |
| SRA |
SRX11548394 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5471118_clone1.BS.ABBS_primer.bw |
5.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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