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| Status |
Public on Oct 01, 2017 |
| Title |
K1 dmMLE-HR replicateA |
| Sample type |
SRA |
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| Source name |
S2
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| Organism |
Drosophila melanogaster |
| Characteristics |
expression vector: dmMLE-HR
clip antibody: Anti-FLAG M2 Magnetic Beads (M8823 Sigma)
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cells are rinsed with PBS and crosslinked with 0.15mJ/cm2 UV-C light. After crosslinking, the cells are pelleted by centrifugation, snap-frozen in liquid nitrogen and kept at -80˚C until use. Cells are lysed with 0.5mL of lysis buffer, mildly sonicated and immunoprecipitated with anti-FLAG beads for 1hr at ˚C. The beads are washed with lysis buffer and bound material is eluted with 3xFLAG peptide (250µg/mL). The eluate is then incubated with MyONEC1 beads to collect biotinylated target protein, after which the beads are washed with high-stringency buffers (0.1%SDS, 1M NaCl, 0.5% LiDS, 0.5M LiCl and 1% SDS, 0.5M LiCl) to aggressively remove non-specific interactors. 3’-linkers are then ligated with T4 RNA Ligase 1, excess adapters are washed away and 3’-tagged, cross-linked RNA is released with proteinase K digestion and column purification (Zymo DNA Clean & Concentrator).
Reverse transcription is carried out with SuperScript III and barcoded reverse-transcription primers. After reverse transcription, relevant samples are mixed and the cDNA is separated on a 6% 6M Urea PAA gel. Size fractionated cDNA is then processed essentially as described in the iCLIP protocol to generate sequencing libraries (König, J. et al. iCLIP--transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. J. Vis. Exp. (2011). doi:10.3791/2638). uvCLAP reads contain two custom barcodes adjacent to the 5'- and 3'-adapters. The 5 nt 5'-barcodes are flanked by 5 random nt according to the pattern NNNTTTTTNN (N: random nucleotide, T: barcode nucletide) and specify the pulldown condition. The 5 nt semi-random 3'-barcodes DRYYR and DYRRY (D: not C, R: purine, Y: pyrimidine) are used to distinguish the two replicates of each experiment. The 5'-barcodes are located at the first 10 nucleotides of the first mate reads. The read pairs align forward/reverse, thus the reverse complement of the 3'-barcodes is located at the first 5 nucleotides of the second mate reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
5’-barcode: TCACG, 3’-barcode: RYYR
K1_dmMLE-HR_combined_dm3.bed
K1_dmMLE-HR_combined_dm3.bw
K1_dmMLE-HR_jamm_dm3.narrowPeak
K1_dmMLE-HR_peakachu_dm3.gff
K1_dmMLE_HR_combined_gaps_dm3.bed/.bigWig
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| Data processing |
Barcodes and random tags were extracted using custom fastqpl scripts (Horton P. fastapl – a utility for versatile and easy processing of fasta format data (and fastqpl for fastq files). F1000Posters 2011, 2:1197 (poster)).
Adapters were trimmed and the library was demultiplexed using Flexbar (v2.32).
Possible readthroughs into the barcoded regions were removed by clipping 10 and 5 nt from the 3’-ends of first and second mate reads.
Reads were mapped to dm3 using bowtie2 (v2.2.0) with parameters --very-sensitive --end-to-end --no-mixed --no-discordant and –maxins=200, only uniquely mapped reads were kept.
Uniquely mapped reads were used to determine crosslinking events as previously described (Ilik, I. A. et al. Tandem Stem-Loops in roX RNAs Act Together to Mediate X Chromosome Dosage Compensation in Drosophila. Mol. Cell 51, 156–173 (2013))
Crosslinking events from mid (M) and low (L) size fractions were combined for each biological replicate.
Combined crosslinked nucleotides of both replicates, extended by 5 nt up- and downstream, were used to create binding profiles in bigWig format.
Peaks were called with JAMM (version 1.0.7rev1, parameters "-d y -t paired -b 50 -w 1 -m normal") on the two replicates for the respective pulldown condition as foreground and the eGFP pulldown condition as background.
Peaks were called with PEAKachu (version 0.0.1alpha2, parameters "--pairwise_replicates -m 0") on the two replicates for the respective pulldown condition as foreground and the two replicates of eGFP pulldown condition as background. All crosslinked nucleotides were extended by 5 nt up- and downstream prior to peak calling to enable shape-based detection of candidate peaks.
Gapped alignments were determined using HMMSplicer (v 0.9.5) with parameters "runHMM.py -g dm3 -c FALSE -w 5 -k 2000 -d TRUE -r FALSE -p 8" for each size fraction of each replicate.
PCR duplicates were removed from HMMSplicer alignments using random barcodes. For multiple alignments at the same position with similar random barcodes (Hamming distance < 2), only the alignment with highest HMMSplicer score was kept.
The resulting alignments of size fractions and replicates were combined and filtered to retain only gapped alignments that do not correspond to canonical splice sites.
The gaps between the split-reads were extracted using a custom script.
Genome_build: dm3
Supplementary_files_format_and_content: bed files of crosslinking events with random tag in name column and number of merged PCR-duplicates in score column. bed files combining crosslinking events of size fractions and replicates. bigWig profiles of combined crosslinking events. JAMM peaks in narrowPeak format. PEAKachu peaks in gff format. Bed files of gapped alignments (gaps_dm3.bed/gaps_dm3.bigWig).
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| Submission date |
Oct 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Asifa Akhtar |
| E-mail(s) |
akhtarlab_data@ie-freiburg.mpg.de
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| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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| Department |
Chromatin Regulation
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| Lab |
Akhtar Lab
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| Street address |
Stuebeweg 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL16479 |
| Series (1) |
| GSE87792 |
A mutually exclusive stem loop arrangement in roX2 RNA is essential for X chromosome regulation in Drosophila |
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| Relations |
| BioSample |
SAMN05893582 |
| SRA |
SRX2236110 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2340382_K1_L_dmMLE-HR_repA_dm3.bed.gz |
465.6 Kb |
(ftp)(http) |
BED |
| GSM2340382_K1_M_dmMLE-HR_repA_dm3.bed.gz |
478.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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