GSM5351805: 256_cell_dnmt1_mKD_total_RNA_seq_rep2; Danio rerio; RNA-Seq
The embryos were dechorionated manually by tweezers and transferred into 750 µl Trizol (Invitrogen, Cat 15596018). About 10 fresh embryos were transferred into Trizol and vortexed until no visible particles. 150 µl chloroform (Amresco, Cat 0757) was added and mixed thoroughly. The mixture was then transferred into phasemaker tube (Invitrogen, Cat A33248) and spun at 14,000 rpm for 15 min. Next, the top phase was taken out from the tube, added 1 µl LPA (Sigma, Cat 56575) and mixed well using pipettes. Then, RNA was precipitated by adding 750 µl isopropanol (Sigma, Cat 59304) at -20℃ overnight. At next day, the tube was spun at 14,000 rpm for 30 min and supernatant was removed. The pellet was washed with freshly 70% ethanol, re-suspend in 20 µl RNase-free water and stored at -80℃ for later usage. NEBNext rRNA Depletion Kit (NEB, Cat E6310S) was used to deplete ribosomal RNA according to the manufacture's instruction. Briefly, rRNA were hybridized with probes and digested with RNase H, then excess probes were digested with DNase I. After that, NEBNext RNA sample purification beads were used to purify rRNA depleted RNA. Purified RNA was fragmented before cDNA synthesis at 95℃ for 8 min. Double-stranded cDNA was synthesized with NEB Next first-strand (NEB, Cat E7771S) and second-strand synthesis modules (NEB, Cat E7550S), then purified with Ampure XP beads (Beckman, Cat A63882). Synthesized cDNA was subjected to library preparation with NEBNext Ultr II DNA Library Prep Kit (NEB, Cat E7645S). DNA was end-repaired, adenylated, and ligated to TruSeq sequencing adaptors. DNA were amplified using KAPA HF HotStart ReadyMix (KAPABiosystem, Cat RR2602). The amplified DNA was size-selected using Ampure XP beads for 200-500 bp DNA fragments. All libraries were sequenced by Illumina Hi-Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.