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Sample GSM5513488 Query DataSets for GSM5513488
Status Public on Oct 26, 2021
Title RNA-K27M_KrimpRNAi_35230_2
Sample type SRA
 
Source name 3rd instar larvae eye discs
Organism Drosophila melanogaster
Characteristics developmental stage: 3rd instar larvae
tissue: eye-antennal imaginal discs
genotype: K27M KrimpRNAi_35230
Extracted molecule total RNA
Extraction protocol RNA was extracted from Drosophila 3rd instar larvae eye discs using a Qiagen kit followed by DNAse treatment. For smRNA-seq RNA was extracted from Drosophila 3rd instar eye discs using Qiagen kit with Trizol, spike-in controls added at the beginning of the protocol, and DNAse treatment was added at the end. For crosslinked ChIP, cells were crosslinked (1% formaldehyde, 10mins) and sonicated. Protein:DNA complexes were immunoprecipitated with specific antibodies pre-bound to protein A or goat anti-mouse IgG magnetic beads. Eluted DNA underwent crosslink reversal, and were purified using Qiagen PCR purification columns. For RNA extraction, 1 million TEX cells were pelleted, washed with PBS and resuspended in 350ul RLT buffer (Qiagen-RNAeasy mini kit) with beta-mercaptoethanol. The suspension was then stored at -80oC until frozen. Sample was thawed and RNA extraction was performed using Qiagen's RNAeasy mini kit as per the manufacturer's instructions.
total RNA samples submitted to Genome Quebec facility for quality control assessment and library preparation. Illumina HiSeq 4000 with miRNA size select has been used for smRNAseq and Illumina HiSeq 4000 using mRNA stranded library has been used for RNA-seq. The eluted ChIP DNA undewent adaptor ligation based library construction for single-end 50bp Illumina sequencing on HiSeq 4000. RNA-seq library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts, paired-end sequencing (100 bp) was performed on the Illumina HiSeq 2500 or 4000 platform.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Data processing For smRNA samples: smRNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor, other Illumina-specific sequences and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 15 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genome (dm6) using novoalign (v3.02.12) with a score difference of 0 for calling multiple mapping (-R 0), reporting a maximum of 100 alignments per read (-r All 100), setting miRNA mode (-m 100) and using -l 14 and -t 10,5.5 as alignment scoring parameters.
For RNA samples: RNA sequencing reads were trimmed using Trimmomatic (v0.32), removing adaptor and other Illumina-specific sequences as well as the first four bases from the start of each read, and low-quality bases at the end of each read, using a 4bp sliding window to trim where average window quality fell below 30 (phred33 < 30). Trimmed reads with less than 30 bases were discarded. The resulting clean set of reads were then aligned to the drosophila reference genomes (dm6) using STAR (v2.3.0e) with default parameters. Reads mapping to more than 10 locations in the genome (MAPQ < 1) were discarded.
for ChIP samples: For ChIP-seq data, reads were trimmed of TruSeq adaptor sequences using trimmomatic. Trimmed reads were then aligned to dm6 using BWA-MEM, PCR duplicates were soft-clipped using Picard. BigWig files were generated using Homer, and consist of uniquely aligned reads without PCR duplicates.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig files were generated by bedtools (v2.27.1) genomeCoverageBed, using the bam generated by STAR (RNA files) or Novoalign (smRNA files) and a normalization factor equal to the number of mapped reads divided by 100 million. bedGraph files were generated by HOMER, using the bam generated by bwa-mem.
 
Submission date Aug 11, 2021
Last update date Oct 26, 2021
Contact name Claudia L Kleinman
E-mail(s) claudia.kleinman@mcgill.ca
Phone 514-340-8222 26129
Organization name Lady Davis Institute for Medical Research
Department Human Genetics
Street address 3999 Côte Ste-Catherine Road
City Montréal
State/province Québec
ZIP/Postal code H3T 1E2
Country Canada
 
Platform ID GPL25244
Series (1)
GSE140979 K27M and K36M mutations on non canonical Histone 3.3 promote redistribution of antagonistic chromatin marks, disrupted development, and tumorigenesis
Relations
BioSample SAMN20708695
SRA SRX11716850

Supplementary file Size Download File type/resource
GSM5513488_RNA-K27M_KrimpRNAi_35230_2.sorted.bw 81.2 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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