title: GSM8372103: Ac, m1A-seq-TGIRT-IP-2; Amphidinium carterae; OTHER
library_construction_protocol: Total RNA was extracted from A. carterae using RNAzol®RT (MRC) according to the manufacturer's protocol. Poly(A)+ RNA of A. carterae was isolated using VAHTS mRNA Capture Beads (Vazyme Biotech, Nanjing, China), fragmented at 70 °C for 10 minutes using RNA fragmentation reagent (Invitrogen) in a 10 µL reaction mixture and purified with the RNA Clean & Concentrator-5 kit (Zymo Research). For m1A-seq-TGIRT, 25 µL of prewashed Protein A/G Dynabeads were incubated with 3 µL of anti-m1A antibody (MBL #D345-3) at 4 °C for two hours. Subsequently, 500 ng of fragmented mRNA was used for immunoprecipitation, with 50 ng of purified mRNA taken as input. After digestion with Proteinase K, the RNA was purified using RNA Extraction Reagent (Shanghai Acmec Biochemical Co., Ltd, China) and then subjected to library construction. Briefly, the purified mRNA was treated with Antarctic Phosphatase (NEB) and T4 PNK (NEB) at 37 °C for 30 minutes, followed by purification using the Oligo Clean & Concentrator kit (Zymo Research). The dephosphorylated RNA was then ligated to the 3'-RNA adaptor (/5'Phosphate/AGAUCGGAAGAGCGUCGUG/ddC) using T4 RNA Ligase I (NEB) at 23°C for two hours, purified by Oligo Clean & Concentrator again, and reverse transcribed using TGIRT-III (Haigne, China) with the RT primer (5'-ACACGACGCTCTTCCGA-3') in RT reaction buffer (50 mM Tris–HCl pH 8.3, 75 mM KCl, and 3 mM MgCl2) at 57 °C for two hours. The reaction was terminated with 25 mM EDTA and RNA was degraded with 150 mM NaOH, heated to 70 °C for 12 minutes, and purified with Oligo Clean & Concentrator kit. The 5'-DNA adaptor (/5'Phosphate/AGATCGGAAGAGCACACGTCTG/ddC) was ligated using T4 RNA ligase I (NEB) at 23 °C overnight. The single-stranded cDNA was then amplified by PCR using specific primers (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3', NNNNNN denotes the 6-bp barcode sequence), and the product was purified with AMPure XP beads (0.9×). Both input and IP samples were sequenced by GenePlus (Shenzhen, China) on DNBSEQ-T7 platform with paired-end 150 bp mode.