title: GSM5230889: MM-seq_T7E1_Ecoli; Escherichia coli; OTHER
library_construction_protocol: Chlamydomonas reinhardtii: cells were treated with lysis buffer (50mM Tris-HCl(pH8.0), 200mM NaCl, 20mM EDTA, 2% SDS, 1mg/ml Proteinase K) and CTAB buffer (50mM Tris-HCl (pH8.0), 20mM EDTA, 1.4M NaCl, 2% CTAB, 1% PVP4000) at 65℃ for 30 min. Total RNA was digested using 100 µg/ml RNase A for 20 min at room temperature. Genomic DNA was isolated by phenol-chloroform extraction and ethanol precipitation. E. coli: Genomic DNA was extracted using FastPure Bacteria DNA Isolation Mini Kit (Vazyme, DC103). Cell lines: Cells were collected and then lysed by lysis buffer (0.1% TritonX-100, 0.1% Tween-20 diluted in 1× PBS) supplemented with 5 μg Proteinase K (Sangon) and 2 μl RNase A (Takara) under the condition of shaking (~1000 rpm) at 37 ℃ for 30 min. E. coli: MinElute PCR Purification Kit (QIAGEN, 28006) was used to isolate genomic DNA. During MM-seq with endonuclease digestion, 6mA immunoprecipitation reaction was conducted in total volume of 500μL with 4μg fragmented gDNA, 5μL (0.5mg/mL) anti-m6A monoclonal antibody (CST), 100μL 5×IP Buffer, 30μL pre-block protein-A magnetic beads. The mixture was transferred to head-over-tail rotating wheel to incubate at 4 °C overnight and placed on a magnetic rack until beads are captured. The supernatant was discarded and the beads was washed with 500 μL ice-cold 1×IP Buffer by gentle vortex. The beads were re-suspended in the T7E1 or MBN digestion system (M0302S or M0250S, NEB) and incubated for 30 min at 37 °C. After washed with 500 μL ice-cold 1×IP Buffer, 5μL Proteinase K was added to the beads and incubated for 2h at 65 °C with 1,000 rpm. The products were purified by Zymo Research oligo Clean & Concentrator and eluted in 12 μL ddH2O. NGS libraries were constructed for products from genomic DNA using NEBNext Ultra II DNA Library Prep Kit (E7645S). Same procedure about 6mA immunoprecipitation reaction was applied when using exonuclease cleavage in MM-seq. The beads with IP products were conducted primer extension, end-repair, adaptor ligation and primer extension steps using T4 DNA polymerase (M0203S, NEB), T4PNK (T4 Polynucleotide Kinase, M0201S, NEB), T4 DNA ligase (M0202S, NEB) and Phi29 DNA polymerase (M0264S, NEB), respectively. The beads were washed by 500 μL ice-cold 1×IP Buffer after each treatment. The beads were resuspended in the lambda exonuclease (M0262S, NEB) digestion system incubating for 30 min at 37 °C. After washed by 1×IP Buffer, the beads were treated by 5μL Proteinase K to digest antibody. The mixture were incubated for 2h at 65 °C with 1,000 rpm speed. Then the beads were spin down briefly and placed the tube on magnetic rack for 30s until beads were captured and transferred the supernatant in a new tube. The products were purified by Zymo Research oligo Clean & Concentrator and elute in 12 μL ddH2O. After primer extension by Phi29 DNA polymerase, the products were added into R2 Adapter ligation reaction. Then, the products were clean up by using 52 μL well-mixed AMPure beads and eluted dsDNA fragment in 30 μL ddH2O. After the PCR amplification, the library was purified using 50 μL well mixed AMPure beads or agarose gel size selection.