biostudies-arrayexpressAntonio LentiniGallus gallushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14443Sex-chromosome dosage compensation 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.biostudies-arrayexpressNucleic Acid Extraction - RNA extraction was performed using Qiagen's RNeasy Plus kit according to the manufacturer's instructions. RNA concentration was measured on a Nanodrop 2000 instrument.Library Construction - RNA-seq library preparation was performed using Illumina's TruSeq RNA Library Prep Kit v2 according to the manufacturer's instructions. Briefly, the samples were incubated at 65°C for 5 min, followed by bead purification to separate and elute polyA RNA-bound beads. The eluted RNA was incubated at 94°C for 8 min followed by 4°C on hold to elute, fragment and prime the RNA for first-strand synthesis. First strand synthesis was performed by adding 8 ul of first-strand master mix (containing 1ul of Superscript II reverse transcriptase for each 9 ul First Strand Master mix) to each sample and running the following program: 25°C for 10 min, 42°C for 50 min, 70°C for 15 min and hold at 4°C. For second strand synthesis, 25 ul of second strand master mix was added to each sample, followed by incubation at 16°C for 1h. After AMPure XP bead purification, end repair was performed by adding 40 ul End Repair Mix to each sample and incubating at 30°C for 30 min. After bead purification, the 3’ ends were adenylated by adding 12.5 ul A-tailing mix to each sample followed by incubation at 37°C for 30 min, 70°C for 5 min and hold at 4°C. Indexing adapters were ligated by adding 2.5 ul Ligation mix and 2.5 ul RNA adapter Index (unique to each sample) per sample and the mixture was incubated at 30°C for 10 min followed by addition of 5 ul of Stop Ligation buffer to each sample to stop the ligation reaction. After bead purification, DNA fragments were enriched through PCR by adding 5 ul PCR primer cocktail and 25 ul PCR Master Mix to each sample. The reaction was performed using the following program: 98°C for 30s, 15 cycles of 98°C for 10s, 60°C for 30s, 72°C for 30s followed by 72°C for 5 min and on hold at 10°C. Finally, the libraries were bead-purified using AMPure XP magnetic beads.Sequencing - e library pool was sequenced on a Nextera Nextseq 550 using a Nextseq 500/550 High-Output 75 cycle sequencing kit v2.5 with the following settings: Read 1 = 72 cycles, Index 1 = 10 cycles, Index 2 = 10 cycles.Sample Collection - Pellets of approximately 1 million chicken embryonic fibroblasts were pelleted at 300g for 5min and the supernatant was removed. To eliminate serum-containing media, the pellets were washed twice in 1x PBS before a final centrifugation at 300g for 5min and supernatant removal.Sequencing - Library quality and size was validated by running the samples on a High Sensitivity dsDNA Bioanalyzer chip and quantified by real-time quantitative PCR (RT-qPCR). 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 = 76 cycles, Index 1 = 6 cycles, Index 2 = 6 cycles.Sample Collection - Brain, kidney, liver, skin, ovaries and testes were isolated from adult chickens.Library Construction - Cell lysis. Bulk Smart-seq2 was performed as previously described [Larsson et al. 2019] with slight modifications. Specifically, 2ng of purified tissue RNA was added to 3μl of Smart-seq2 lysis buffer (1uM oligo-dT primer [5Biosg//idSp//idSp//idSp/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN; IDT], 0.5mM (each) dNTPs, 0.2% Triton-X-100, 1 U/μl RNase inhibitor [Takara]. To ensure RNA denaturation, the samples were incubated at 72°C for 3 minutes and immediately placed on ice. Reverse transcription and cDNA synthesis: 5.7 μl of reverse transcription mastermix (1x Superscript II first-strand buffer, 5mM betaine [Sigma], 6mM MgCl2 [Ambion], 1uM TSO [5’- Biotin-AGAGACAGATTGCGCAATGHHHHHHrG+GG-3’; IDT], 1.7U/μl of recombinant RNase inhibitor [Takara], 17U/μl Superscript II reverse transcriptase) was added to each sample and the reaction took place as follows: 42°C for 90min, 10 cycles of 50°C for 2 min, 42°C for 2 min, followed by, 70°C for 15 min and 4°C on hold. For the pre-amplification PCR, 15μl of PCR mastermix was added to each sample (1X Kapa HiFi HotStart ReadyMix [Roche], 0.1uM forward primers [5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG-3’; IDT] and 0.1uM reverse primers [5’-ACGAGCATCAGCAGCATACGA-3’, IDT]) and the reaction took place as follows: 98°C for 3 min, 8 cycles of 98°C for 20 sec, 67°C for 15 sec, 72°C for 6 min, and followed by 72°C for 5 min, and 4°C on hold. cDNA purification. Purification of the cDNA libraries was performed by combining the cDNA samples to 22% PEG magnetic beads or AMPure XP beads at a ratio of 1:0.8. Briefly, the mixture was incubated at room temperature for 8 minutes and on the magnetic rack for 5 minutes. The supernatant was removed and the bead pellet was washed twice with freshly-prepared 80% ethanol. The bead pellet was left to air-dry for 3 minutes and the cDNA libraries were eluted in 17μl of EB buffer [Qiagen]. Quantification of the cDNA libraries was performed using the Quantifluor dsDNA kit [Promega]. The libraries were normalised to 1ng/ul. Tagmentation. 2ng of cDNA was combined with 18μl of tagmentation mix containing 10mM TAPS-NaOH [Sigma], 5mM MgCl2 [Thermofisher], 8% PEG-8000 and 0.5 μl of in-house produced Tn5 at 44.5uM. The samples were incubated at 55°C for 8 minutes. To strip the Tn5 off the cDNA, 3μl of 0.2% SDS solution was added to each sample and the mixture was incubated at room temperature for 5 minutes. PCR amplification. 1.5μl of combined Nextera i7 and i5 [IDT] were added to each sample as well as 25 μl of PCR mastermix (1x KAPA HiFi PCR buffer, 0.06mM (each) dNTPs, 1U KAPA HiFi polymerase). The reaction took place as follows: 72°C for 3min, 95°C for 30 sec, 10 cycles of 95°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 libraries were pooled and purified as described above. The final concentration of the pool was measured on a Qubit 4.0 using the Qubit dsDNA High Sensitivity Assay kit [Molecular probes] and the library fragment distribution was inspected on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA chips. ThNucleic Acid Extraction - RNA isolation was performed using the phenol-chloroform extraction method. Briefly, 50-100mg of tissue was isolated and homogenised using 1 ml of TRIzol reagent [Thermofisher] and a tissue homogeniser. To precipitate the RNA, 500μl of isopropanol was added per 1 ml of TRIzol and the mixture was incubated for 10 minutes followed by centrifugation at 12000 rpm at 4°C for 10min. To wash the RNA, the pellet was resuspended in 1 ml of 75% ethanol per 1 ml of TRIzol used and centrifuged for 5 min at 7500 rpm and 4°C. The supernatant was discarded and the pellet left to air-dry for 5-10 min. The pellet was resuspended in 50μl of nuclease-free water, incubated at 55°C for 15 min and stored in -80°C. The concentration was measured using a Nanodrop 2000 instrument.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Raw multiplexed RNA-seq data was aligned and quantified to the GRCg6a genome (Gallus_gallus.GRCg6a.dna_sm.toplevel.fa.gz + Gallus_gallus.GRCg6a.100.gtf transcript annotations) using zUMIs (v.2.9.4c, - UMI(12-19), find_pattern: ATTGCGCAATG, additional_STAR_params: ‘--clip3pAdapterSeq CTGTCTCTTATACACATCT’)[REF] with barcode- and UMI filtering cutoffs allowing 1 base at phred 20 using a list of expected barcodes for edit distance-based binning.Data Transformation - For demultiplexed and aligned bam files, read groups were added and samples were merged according to genotype using GATK ( AddOrReplaceReadGroups, MergeSamFiles [gatk --java-options “-Xmx128G”])[978-1-4919-7519-0]. Variants were called using bcftools (v.1.10.2) mpileup (--max-depth 8000 --skip-indels) and call (-mv, in ploidy mode)[10.1093/gigascience/giab008] then filtered for a depth over 5 reads with an allele frequency >50% using bcftools filter (-i ‘DP>5 & AF>0.5 & QUAL>10’). Next, “unique WL” variants were subsetted from RJF variants using bcftools isec (-C -w 1) and “common RJF” variants were subsetted from the “unique WL” list. As a 2nd pass filtering, allelic expression was quantified for “unique WL” variants and variant-level count tables were calculated using zUMIs with a standard GRCg6a reference genome (see below) and variants with an agreement with chicken strain in <50% of males or females were excluded to form the “final WL” variant list. Next, a custom GRCg6a reference genome was created by first inserting “common RJF” variant bases to correct for strain deviations using bcftools consensus (v.1.10.2) followed by N-masking using “final WL” variants using bcftools consensus (v.1.10.2, --mask). A STAR index was created using the WL N-masked GRCg6a genome and used for zUMIs alignment and quantification. Allelic quantification was performed on the zUMIs output bam files as previously described [Larsson Nature, Lentini Nat Comms]. Briefly, variants were mapped to transcriptome positions and intersected with bases overlapping N-masked positions of the genome using the CIGAR string and reads were assigned to RJF/WL genotypes if >0.66 of basecalls matched the genotype and allelic read counts were summarised per gene and cell.UnknownTranscriptomicsGenomicsProteomicsNextSeq 550RNA-seq of coding RNAGallus gallusAntonio LentiniBjörn ReiniusNatali PapanicolaoufalseMulti-layer dosage compensation of the avian Z chromosome (bulk RNA-seq)Sex-chromosome dosage compensation 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.2025-06-29T00:00:00Z2024-09-10T13:07:12.893Z2024-09-10T13:07:12.893ZE-MTAB-14443ERP164011E-MTAB-14393E-MTAB-14392E-MTAB-14391E-MTAB-14390EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184