title: Single-cell bisulfite sequencing of fat-tailed dunnart (Sminthopsis crassicaudata) E9 pooled embryos
design: Single-cell bisulfite sequencing of fat-tailed dunnart (Sminthopsis crassicaudata) E9 pooled embryos
library_construction_protocol: Tissues were collected from a long term breeding colony of fat-tailed dunnarts at Macquarie University. All animal work was conducted with approval from the Macquarie University Animal Ethics Committee (ARA: 2020/019). Staged embryos were collected by daily weighing of paired females through the breeding season to identify the weight changes associated with ovulation. Once at the right day of gestation, females were humanely killed with an intra-peritoneal injection of Sodium Pentobarbitone. Following dissection, embryos were snap frozen in liquid nitrogen for downstream processing. Single nuclei from 3 snap frozen E9 embryos were isolated using The S2 Genomics Singulator Platform, with the Low Volume Nuclei Isolation V2 protocol according to manufacturer instructions. DAPI-positive nuclei were sorted in 96-well plates containing 2.5μL of Buffer RLT (QIAGEN) and 1U/µL RNAse inhibitor using BD FACSAria™ III Cell Sorter. Plates were immediately placed on dry ice, sealed, and stored at -80°C until library preparation. Single cell bisulfite sequencing (scBS-seq) libraries were prepared as described with minor modifications (Clark et al Nature Protocols 2017). First, RNA was removed from single cell lysates using oligo-dT conjugated magnetic beads. Genomic DNA was then purified using a 0.8X volume of AMPure XP beads (Beckman Coulter) and subjected to bisulfite conversion using the EZ Methylation Direct Kit (Zymo). The oligo used in preamplification was modified to be compatible with NEBNext index primers used later in the protocol, and to prevent self-amplification: /5SpC3/CTACACGACGCTCTTCCGATCTNNNNNN (IDT, HPLC purified). After completing 5 rounds of preamplification and exonuclease treatment, the first-strand synthesis products were cleaned-up using a 0.65X volume of AMPure XP beads. Adaptor 2 tagging was then performed, and a 0.65X volume of buffer from AMPure XP beads was used to purify the second-strand synthesis products. Library amplification was then performed using NEBNext Multiplex Oligos for Illumina (Dual Index Primers) and 1x NEBNext Q5 Ultra II Master Mix (New England Biolabs), as follows: 98°C for 30 s; 14 repeats of 98°C for 10 s, 65°C for 30 s, 65°C for 45 s; 65°C for 5 min; 4°C hold. Amplified scBS-seq libraries were cleaned-up using a 0.65X volume of buffer from AMPure XP beads, assessed using the TapeStation High Sensitivity D5000 ScreenTape assay (Agilent), and quantified using Qubit dsDNA High Sensitivity assay (Thermo Fisher Scientific)