Biotinylated primers

Library preparation was performed using a mosquito robot HV genomics (TTP Labtech Ltd, UK) following the Smart-seq2 protocol^{40 (link)}. Briefly, 384 well plates containing sorted single nuclei in lysis buffer were thawed and reverse transcription with Superscript II (Life Technologies, cat #: 18064014) and PCR using KAPA Hifi HotStart ReadyMix (Kapa cat #: KK2602) were performed with the following biotinylated primers (Qiagen): Oligodt (AA GCA GTG GTA TCA ACG CAG AGT ACT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTV N), TSO (AAG CAG TGG TAT CAA CGC AGA GTA CATr GrG+G) and ISPCR primers (AA GCA GTG GTA TCA ACG CAG AGT). Following RT-PCR, clean up with Agencourt AMPure XP beads (Beckman Coulter cat #: B37419AA) was carried out and sample concentrations were measured using Bioanalyzer (Agilent Technologies) and normalized at a concentration of 0.3 ng/µl. The Nextera XT DNA library prep kit (Illumina cat #: FC-131-1096) was used for subsequent sample preparation. Samples were subjected to a tagmentation reaction, indexing, and PCR amplified. Libraries were then mixed in 384-sample pools and purified with Agencourt AMPure XP beads. Ready DNA libraries were quality controlled using D1000 Screen Tape Assay (Agilent Technologies). Samples were sequenced at the Functional Genomics Center Zurich on Illumina HiSeq 2500 or HiSeq4000 sequencers with single-end 125bp reads.

1 μg DNA was subjected to bisulfite treatment using EZ DNA methylation-Gold kit (Zymo Research) following the manufacturer’s protocol. For pyrosequencing-based methylation analysis of CpG76, the bisulfite modified DNA was PCR amplified using the biotinylated primers (Qiagen) shown in table 1. The biotinylated PCR products were purified using the sepharose beads and denatured using 0.2 M NaOH solution using the pyrosequencing vacuum prep tool. Subsequently, 0.3 μM pyrosequencing primer (Table 1) was annealed to the purified single stranded PCR product and sequencing was carried out using MD96 pyrosequencing System (Qiagen). Quantitation of cytosine methylation was carried out using the PyroMark96 software. The methylated and unmethylated human control DNA (Qiagen) were used to verify bisulfite conversion.