{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Antonio Lentini"],"organism":["Gallus gallus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14391"],"description":["Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZZ/ZW system) have been found to lack full compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency, similar to mammalian X-upregulation, and that Z-protein levels are balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrates a remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Genomic DNA was extracted from frozen CEF pellets of approx 1 million cells using the Monarch Genomic DNA purification kit according to the manufacturer’s instructions. Briefly, the frozen cell pellets were slowly thawed on ice and resuspended in 100 ul ice-cold PBS. 1ul Proteinase K and 3ul RNase A were added to the samples and mixed by vortexing to ensure digestion of remaining protein and RNA respectively, followed by 100ul cell lysis buffer to release the genomic DNA and incubation for 5min at 56°C with agitation at 1400 pm. To bind and elute the genomic DNA, 400ul gDNA Binding buffer to each sample and the samples were thoroughly mixed by pulse-vortexing. The lysate was then transferred to a gDNA purification column and centrifuged at 1000g for 3min and at 12000g for 1min to clear the membrane. The membrane-bound gDNA was washed twice with 500ul gDNA wash buffer by centrifuging at maximum speed for 1 min. gDNA was then eluted by adding 50ul preheated gDNA elution buffer and concentration was measured at a Nanodrop 2000 instrument.","Sample Collection - Chicken embryonic fibroblasts were cultured to approximately 80% confluency and pellets of 1 million cells were collected by centrifugation at 300g for 5 min. To remove as much of medium and FBS residue as possible, the pellets were washed 3 times in 1x PBS. After a final centrifugation, the supernatant was removed and pellets were placed in -80°C until DNA isolation.","Sequencing - Library fragment size was assessed using a Bioanalyzer high-sensitivity dsDNA chip and library concentrations were quantified using Qubit’s high-sensitivity dsDNA quantification kit on a Qubit 3.0 Fluorometer. Libraries were pooled in equimolar amounts and sequenced on a Nextseq 550 instrument using  a Nextseq 500/550 High-Output 75 cycle sequencing kit v2.5 [20024906] with the following settings: Read 1 = 74 cycles, Read 2 = 74 cycles,  Index 1 = 10 cycles, Index 2 = 10 cycles.","Library Construction - To prepare for DNA-seq library construction, gDNA was diluted to a final concentration of 1ng/ul. gDNA tagmentation was performed using an in-house prepared Tn5 enzyme as previously described (Picelli et al. 2014). Briefly, 5ng of gDNA was incubated with 15ul tagmentation master ix (10mM TAPS, 5mM MgCl2, 10% DMF, 2.25uM Tn5)at 55°C for 8min. To strip the Tn5 from the DNA, 3.5ul of freshly-prepared 0.2% SDS solution was added to each reaction and the samples were quickly centrifuged and incubated at room temperature for 5min. The samples were indexed using 2.5ul of 1uM pre-mixed Nextera index primers and post-tagmentation PCR was performed by adding 16.5 ul PCR master mix (1x KAPA HiFi PCR buffer, 0.6mM (each) dNTPs, 1U/ul KAPA HiFi polymerase) to each sample and incubating using the following program: 72°C for 3 min, 95°C for 30s, 6 cycles of [95°C for 10s, 55°C for 30s, 72°C for 30s], 72°C for 5min and 4°C on hold. Double purification of the amplified libraries was performed using in-house 22% PEG magnetic beads (Hagemann-Jensen et al. 2019).  22% PEG magnetic beads were combined with the pooled DNA libraries in a bead-to-sample ratio of 0.9:1 and incubated at room temperature for 8 minutes. The samples were then placed on a magnetic rack for 5 minutes. The clear supernatant was then removed and discarded and the bead pellets were washed twice with freshly-prepared 80% EtOH. The beads were left to air-dry for 3 minutes while remaining on the magnetic rack. The samples were eluted in 30ul. In order to ensure complete removal of residual impurities and primer-dimers, the purification was repeated as described above and the final sample was eluted in 17μl of nuclease-free water."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Raw BCL files were converted to FASTQ format using  using bcl2fastq (v.2.20.0.422). Raw DNA-seq data was adapter- and quality trimmed using fastp (v.0.20.0, –adapter_sequence CTGTCTCTTATACACATCT –adapter_sequence_r2 CTGTCTCTTATACACATCT) [10.1093/bioinformatics/bty560] and aligned to the GRCg6a reference genome using minimap2 (v.2.24-r1122, -ax sr)[10.1093/bioinformatics/bty191]","Data Transformation - Reads were sorted, mate-pair information fixed and duplicates marked using biobambam2 (v.2.0.87, bamsort fixmates=1 markduplicates=1). Variants were called using bcftools mpileup (–ignore-RG -a AD,DP, –max.depth 8000) and call (-mv, in ploidy mode) using ZZ and ZW ploidies with sample-sex information. Variants within 5bp of indels were excluded and heterozygous variants sequenced to a depth over 5 reads with a minor allele frequency over 10% were filtered using bcftools filter (-g 5 -i ‘TYPE=”snp” & QUAL >10 & INFO/DP>5 & GT=”het” & MAF>0.1’). To calculate DNA copy numbers, the GRCg6a genome was binned into 100kb bins using bedtools makewindows (v.2.30.0, -w 100000)[10.1093/bioinformatics/btq033] and binned read counts were calculated using bedtools multicov (-q 13). Genome statistics were also calculated for the same bins; mappability (see above) using deeptools (multiBigwigSummary BED-file); nucleotide frequencies using bedtools (nuc); assembly gaps were obtained from UCSC and gaps >1kb were kept and bins within 500kb were identified using bedtools (window -w 500000 -c); RepeatMasker rmsk track was obtained from UCSC and overlapped with bins using bedtools (intersect -wao | map -c 10) and percentage overlap was calculated. Bins with >2.5% N bases or average mappability <50% or within 500kb of a large assembly gap or with a rmsk fraction 2 MADs above median were excluded. Data was corrected for GC-content and mappability and DNA copies were estimated using HMMcopy (v.1.38.0, correctReadcount mappability = 0.8). Expected ploidies were set to 2 for diploid samples and 3 for triploid and multiplied with DNA copies to adjust for ploidy and regions annotated as ideal by HMMcopy were used for plotting. For base-resolution variants, only variants with a read depth of 6-50, heterozygous genotype and >0 variance were kept and variants overlapping excluded genome bins were removed."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 550"],"study_type":["DNA-seq"],"species":["Gallus gallus"],"pubmed_authors":["Antonio Lentini","Björn Reinius","Natali Papanicolaou"],"additional_accession":[]},"is_claimable":false,"name":"Multi-layer dosage compensation of the avian Z chromosome (DNA-seq)","description":"Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZZ/ZW system) have been found to lack full compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency, similar to mammalian X-upregulation, and that Z-protein levels are balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrates a remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.","dates":{"release":"2025-06-30T00:00:00Z","modification":"2024-08-27T16:47:03.532Z","creation":"2024-08-27T16:47:03.532Z"},"accession":"E-MTAB-14391","cross_references":{"ENA":["ERP163586"],"Biostudies":["E-MTAB-14393","E-MTAB-14392","E-MTAB-14390"],"EFO":["EFO_0002944","EFO_0004170","EFO_0002693","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}