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| Status |
Public on Dec 15, 2016 |
| Title |
fk041_Lmaj_DIP1 |
| Sample type |
SRA |
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| Source name |
Lmaj_DIP
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| Organism |
Leishmania major |
| Characteristics |
pull_down_target: 5hmU
pull_down_method: Antibody (DIP)
antibody info.: polyclonal anti-5-hydroxymethyluridine (Abcam, ab19735, RRID:AB_722498)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA samples for Leishmania major (ATCC 30012D, lot 60685413, lot 63717803, lot ATCC-CUST-30012D) and the RNA sample for Leishmania major (lot ATCC-CUST-30012R) were obtained from ATCC. The genomic DNA sample for Leishmania donovani GR383WT1 (single batch) was provided courtesy of Prof. Charles L. Jaffe (National Center for Leishmaniasis, Kuvin Centre for Study of Tropical & Infectious Diseases, Jerusalem).
5hmU DNA immune-precipitation (DIP)
Fragmented DNA samples were subjected to adapter ligation using NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) and TruSeq sequence adapter solutions (Illumina). For generating 5hmU-enriched library by DIP, the adapter ligated DNA (in 20 uL) was heated to 95 C for 10 min and then immediately cooled on ice to denature. A mixture of the denatured DNA, anti-5-hydroxymethyluridine antibody ab19735 (5 uL, Abcam, RRID:AB_722498), and rabbit anti-goat IgG H&L ab6697 (10 L, Abcam, RRID:AB_955988), and 0.1% (v/v) tween 20 in PBS was incubated with rotation at 4 C, then added to Dynabeads Protein G (Life Technologies). The beads were washed 3 times with 0.1% (v/v) tween 20 in PBS (200 uL), re-suspended in 0.1% (v/v) tween 20 in PBS and treated with proteinase K (20 ug/uL, 1.75 uL) at 50 C for 2 h. The supernatant containing the eluted DNA was collected and purified. The obtained 5hmU enriched libraries were amplified by amplified by PCR. The libraries were sequenced on Illumina MiSeq with single-end read (length of 150bp) or paired-end read (length of 75bp x 2).
5hmU chemical enrichment
DNA samples were ligated with chemically modified Truseq adapters prior to the biotin tagging. The adapter ligated DNA sample was oxidised using the oxidant solution provided in the TrueMethyl kit (Cambridge Epigenetics) followed by desalting with Micro Bio-Spin P-6 Gel Columns (Bio-Rad). The sample was biotinylated by treatment with (+)-biotinamidohexanoic acid hydrazide followed by purification, and used immediately for enrichment.
The biotinylated DNA was bound to Dynabeads MyOne Streptavidin C1 (Life Technologies) by gentle mixing. The beads were placed on a magnetic rack and the supernatant was removed. The beads were washed 5 times and the DNA was eluted by incubating the beads in elution buffer (0.05% (v/v), 5 mM anisidine, 0.1% (v/v) tween 20 in 50 mM sodium phosphate buffer pH 6). The elution process was repeated once more. The obtained 5hmU enriched libraries were purified and amplified by PCR. The libraries were sequenced on Illumina MiSeq (single-end read with length of 150bp).
Base J chemical enrichment
Fragmented DNA sample was treated with NaIO4 in sodium acetate buffer (pH 5.5) at 40 C followed by purification of DNA. The resultant DNA was biotinylated by treatment with (+)-biotinamidohexanoic acid hydrazide followed by purification. The biotinylated DNA was subjected to NGS adapter ligation using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) and TruSeq sequence adapter solutions (Illumina). Downstream procedures for base J enrichment were carried out as described in 5hmU chemical enrichment sequencing except the PCR was carried out while base J modified DNA fragments were bound to the magnetic beads. The libraries were sequenced on Illumina MiSeq (single-end read with length of 150bp).
Total RNA library preparation
Total RNA (1 ug) was fragmented by incubating in a fragmentation buffer (40 mM Tris-HCl pH 8.2, 100 mMLiCl, and 30 mM MgCl2) followed by purification. The RNA was treated in T4 PNK (Thermo Fisher Scientific) in absence of ATP and purified to remove phosphate group at 3 -end. The adapter (5 - 5rApp AGATCGGAAGAGCACACGTCTG-SpC3- 3 ) was ligated to the RNA by T4 RNA ligase 2 K227Q (New England BioLabs), followed by purification. Reverse transcription was carried out using the reverse primer (5 -CAGACGTGTGCTCTTCCGATCT-3 ) and SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) followed by RNA degradation by heating up samples at 95 C in presence of NaO and purification. ssDNA adapter was ligated to the resulted single-stranded DNA, by following literature protocols [1] The obtained samples were amplified by PCR using primers (5 -CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3 and 5 -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3 ). The libraries were sequenced on Illumina MiSeq with paired-end read length 75bp.
Kwok, C. K. & Balasubramanian, S. Targeted Detection of G-Quadruplexes in Cellular RNAs. Angew Chem Int Edit 54, 6751-6754, doi:10.1002/anie.201500891 (2015).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Pull down libraries were sequenced on an Illumina MiSeq with a read length of 151 bp. Reads were trimmed to remove adapters and low quality 3’ ends using cutadapt version 1.7. Trimmed reads were then aligned to the reference genome using bwa mem version 0.7.10. Only primary alignments with mapping quality of 10 or more were retained for further analyses. Alignment filtering and manipulations were performed with samtools version 1.1. See Additional file 1, Table S4 for a summary of the mapping step. Peaks of read enrichment were detected using macs version 2.1.0.20140616. Further analyses were performed by means of bedtools, deepTools, DREME, and custom bash, R and python scripts. Detailed protocols for bioinformatics analysis including statistical analysis are available from the github repository (https://github.com/sblab-bioinformatics/mapping-5hmU-in-Leishmania).
Genome_build: Reference genome for L. major: ftp://ftp.sanger.ac.uk/pub/project/pathogens/Leishmania/major/Current/LmjF_v6.1_20131105/fasta/LmjF_v6.1_all_20131105.fa
Genome_build: Reference genome for L. donovani: ftp://ftp.sanger.ac.uk/pub/project/pathogens/Leishmania/infantum/V5210211/Linfantum.fa
Supplementary_files_format_and_content: Output of MACS2 peak caller in “txt” format reporting positions of enriched regions and their statistical significance
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| Submission date |
Jun 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Dario Beraldi |
| E-mail(s) |
dario.beraldi@cruk.cam.ac.uk
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| Organization name |
Cambridge Research Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL22014 |
| Series (1) |
| GSE83384 |
Genome-wide mapping of 5-hydroxymethyluracil in eukaryote parasite Leishmania |
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| Relations |
| BioSample |
SAMN05253216 |
| SRA |
SRX1847180 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2200475_fk041_Lmaj_DIP1_peaks.txt.gz |
31.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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