biostudies-arrayexpressAntonio LentiniGallus gallushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14470Sex-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 (ZW/ZZ system) have been found to lack efficient 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 further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate 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.biostudies-arrayexpressSample Collection - Chicken embryonic fibroblasts (CEFs) were sorted into 384-well PCR plates [Thermofisher] containing 3μl of lysis buffer (5% PEG-8000 [Sigma], 0.1% Triton-X-100 [Sigma], 0.5 units/μl RNase Inhibitor [Takara], 0.5mM (each) dNTPs [Thermofisher], 1uM oligo-dT primer [5’-Biotin-ACGAGCATCAGCAGCATACGAT30VN-3’; IDT]. Sorting was performed using a FACS Aria II. After sorting, the plates were sealed, briefly centrifuged, and stored in -80°C.Library Construction - n brief, single cells were sorted using a Sony SH800S instrument into provided 384-well plates containing lysis buffer, spun down and stored at -80 °C. Upon submitting plates to Xpress Genomics, robotic automated library preparation was performed.Sequencing - Libraries were pooled in equimolar amounts and sequenced on an S4 lane on a Novaseq 6000 instrument at NGI Sweden.Sequencing - Sequencing was performed on the DNBSEQ G400RS platform (MGI Tech) using App-C Sequencing primers.Library Construction - For reverse transcription, 1μl of reverse transcription master mix (25mM Tris-HCl pH 8.3 [Sigma], 30mM NaCl [Ambion; Thermofisher], 2.5mM MgCl2 [Ambion; Thermofisher], 1mM GTP [Thermofisher], 8mM DTT [Thermofisher], 0.5 units/μl RNAse Inhibitor [Takara], 2uM template-switching oligo [5′-biotin-AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3′; IDT], 2U/μl Maxima H- RT enzyme [Thermofisher]) was added to each sample. Reverse transcription was performed at 42°C for 90min, followed by 10 cycles of 50°C for 2min and 42°C for 2min, and terminated at 85°C for 5min. For PCR pre-amplification, 6μl of PCR master mix (1x KAPA HiFi HotStart Buffer [Roche], 0.3mM (each) dNTPs [Thermofisher], 0.5mM MgCl2 [Thermofisher], 0.5uM forward primer [5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAA-3’; IDT], 0.1uM reverse primer [5’-ACGAGCATCAGCAGCATAC*G*A-3’; IDT], 0.02U/μl polymerase) was added to each sample. PCR pre-amplification was performed using the following thermocycler program: 98°C for 3min, 20 cycles of 98°C for 20 sec, 65°C for 30 sec, 72°C for 4 min, followed by 72°C for 5 min and 4°C on hold. cDNA purification was performed using in-house prepared 22% PEG beads at a beads-to-sample ratio of 0.6:1. cDNA was quantified using the Quantifluor dsDNA kit [Promega]. cDNA was normalised to a final concentration of 100pg/ul. For the tagmentation step, 100pg of cDNA were incubated with 1μl of tagmentation mastermix (0.1μl of tagmentation buffer 4x containing 40mM Tris-HCl pH 7.5, 20mM MgCl2, 20% Dimethylformamide, 0.1μl Amplicon Tagment Mix - Tn5 [Nextera], 0.40μl water) at 55°C for 10 min. To strip the Tn5 from the cDNA, 0.5μl of freshly-prepared 0.2% SDS solution [Sigma] was added to each sample and incubated at room temperature for 5 min. The samples were indexed using 1μl of 1uM in-house, pre-mixed Nextera index primers [IDT] and post-tagmentation PCR was performed by adding 3μl of PCR mastermix (1.4μl Phusion HF 5x buffer, 0.2mM (each) dNTPs, 0.01U/μl Phusion HF polymerase) to each sample. PCR was performed using the following program: 72°C for 3min, 98°C for 3min, 10 cycles of 98°C for 10 sec, 55°C for 30 sec, 72°C for 30 sec, followed by 72°C for 5 min and 4°C on hold. The samples were subsequently pooled and purified using in-house 22% PEG magnetic beads with a ratio of beads-to-sample of 0.7:1.Nucleic Acid Extraction - To ensure cell lysis and RNA denaturation, the plates were incubated at 72°C for 10 min and immediately placed on ice.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Data was preprocessed using zUMIs (v.2.9.7) as described above but with the following modifications: 2 mismatches were allowed in detection of UMI-read patterns, and for barcode and UMIs, 4 and 3 mismatches were allowed, respectively. Additionally, spike-in sequences for the 5’ complex set of molecular spikes were included as mappable sequences (https://raw.githubusercontent.com/sandberg-lab/molecularSpikes/main/fasta_reference/molecularSpikes_complexset_5p.fa). Molecular spikes were extracted from aligned bam files using the UMIcountR package33 (https://github.com/cziegenhain/UMIcountR) and overrepresented spike-ins were removed (>5 barcode or >100 sequences).Data Transformation - Next, cells with less than 10% reads in spike-ins were kept and outliers were detected based on low gene detection (log 3 MADs) or read counts (log 5 MADs) and excluded. Spike-in size factors were calculated and UMIs were normalised using scater/scran (v.1.24.0, computeSpikeFactors, logNormCounts transform = “none”).UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000BGISEQ-500RNA-seq of coding RNA from single cellsGallus gallusAntonio LentiniBjörn ReiniusNatali PapanicolaoufalseMulti-layer dosage compensation of the avian Z chromosome (single-cell RNA-seq)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 (ZW/ZZ system) have been found to lack efficient 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 further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate 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.2025-06-30T00:00:00Z2024-09-26T09:17:23.89Z2024-09-26T09:17:23.89ZE-MTAB-14470ERP164522EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184