title: Multi-layer dosage compensation of the avian Z chromosome (single-cell RNA-seq)
design: Multi-layer dosage compensation of the avian Z chromosome (single-cell RNA-seq)
library_construction_protocol: 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. To ensure cell lysis and RNA denaturation, the plates were incubated at 72°C for 10 min and immediately placed on ice. 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.   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.