title: GSM1627837: Human_normal_blsatoyst_MethylC-seq_3; Homo sapiens; Bisulfite-Seq
library_construction_protocol: For MethylC-seq for MII oocyte, 8-cell embryo, morula, ICM and single blastocyst, about 150 cells were lysed in 5 µL lysis buffer (20 mM Tris, 2 mM EDTA, 20 mM KCl,1 mg/mL protease) for 1.5 h at 56°C and then heat-inactivated for 30 min at 75°C. Genomic DNA from sperm, 6-week fetal and placenta were extracted with QIAamp DNA Mini Kit (Qiagen). Placenta and 6-week fetal RNA were extracted with Direct-zol™ RNA MiniPrep kit (Zymo). Blastocyst RNA were amplified and reverse-transcripted with REPLI-g WTA Single Cell Kit (Qiagen). After adding 45 µL nuclease free water and 0.5% spike-in unmethylated lamda DNA (Promega), the DNA included lysis was then fragmented with Covaris shearing condition of duty cycle 5%, intensity 5, cycles/burst 200 and duration 60s. The lysis was then concentrated to 30 µL for the following operations. First, the fragmented DNA was end-repaired by incubating with 5ul end-repair enzyme mixture ( 3.5 ul T4 DNA ligase buffer (NEB), 0.35 ul 10mM dNTP, 1.15ul NEBNext End Repair Enzyme Mix (NEB)) for 30min at 20°C and then heat-inactivated for 30 min at 75°C. After end-repair, another 5ul of dA-tailing mixture ( 0.5 ul T4 DNA ligase buffer, 1 ul Klenow exo- (NEB), 0.5 ul 100mM dATP and 3ul nuclease free water ) was added to the tube and incubated for 30min at 37 °C followed a heat-inactivation for 30 min at 75°C. Finally, 10 ul ligation mixture (1 ul T4 DNA ligase buffer, 0.5 ul 100 mM ATP, 1.5 ul 50 mM cytosine methylated Illumina adapter, 2 ul T4 DNA ligase (NEB) and 5 ul nuclease free water) was added to the tube and incubated at 16 °C overnight. Bisulfite conversion reaction was performed with the EZ DNA methylation-Gold Kit (Zymo Research) according to the manufacturer’s instructions. 100ng Carrier RNA was added during the bisulfite reaction and column purification. The purified DNA was then amplified with two round PCR with KAPA HiFi HotStart Uracil+ ReadyMix (KAPA). After the first round of 6 cycles PCR, the product was purified with Ampure XP beads (Beckman) to discard the short fragments and adapter-self ligations. Then, another round of 6-8 cycles of PCR was performed to obtain sufficient molecules for sequencing.  RNA-seq Libraries were constructed with NEBNext® Ultra™ RNA Library Prep Kit (NEB) according to manufacture illustrator. QC-passed libraries were then sequenced on Hiseq2000 or Hiseq2500 platform with pair-end module.