title: GSM8652690: A549-hACE2, mock, no treatment, m6A-IP, rep 1; Homo sapiens; RIP-Seq
library_construction_protocol: The m6A/MeRIP-Seq was performed as described with some modifications [Neuron 97, 313-325.e6 (2018)]. Briefly, total RNA was extracted from A549-hACE2 and patient PBMCs using TRIzol (Life Technologies) followed by purification using illustra RNA spin Mini kit (GE Healthcare, UK). mRNA was isolated from 10 μg total RNA using Dynabeads Oligo (dT)25 (Thermo Fisher; 61006) according to manufacturer's instructions. Ten percent of a total of 150 ng mRNA was used as input, while the rest was incubated with 3 ug anti-m6A polyclonal antibody (Synaptic Systems; 202003) that was pre-conjugated to Dynabeads Protein A (Thermo Fisher; 10001D) in 500 uL IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Igepal CA-630) for 2 hours at 4°C. After washing the beads twice with IP-buffer and twice with high-salt wash buffer (50 mM Tris pH 7.4, 500 mM NaCl, 0.1% Igepal CA-630) for 5 minutes each, the m6A-tagged mRNA was eluted using 100 uL IP-buffer containing 6.7 mM N6-Methyladenosine (Sigma-Aldrich; M2780) and 40 U RNase Inhibitor (NEB, M0314S) and then recovered with RNA Clean and Concentrator-5 spin columns (Zymo; R1015). The input and m6A-IPed mRNA were subjected to library generation using the SMART-seq protocol as described [Nature Protocols 9, 171-181 (2014)]. For first strand cDNA synthesis, the mRNA was mixed with 0.25 µL RNase inhibitor and 1 µL CDS primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') and heated to 70 °C for 2 min. Then the mixture containing 0.5 µL of 100 mM DTT, 0.3 µL of 200 mM MgCl2, 1 µL of 10 mM dNTPs, 0.25 µL RNase inhibitor, 1 µL of 10 µM TSO primer (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3'), 2 µL of 5X SMARTScribe RT buffer and 0.5 µL SMARTScribe reverse transcriptase (Takara, 639536) was added to perform reverse transcription. The cDNA was then amplified by Advantage Polymerase Mix (TAKARA, 639201) with IS primer (5'-AAGCAGTGGTATCAACGCAGAGT-3'). After purification with 0.8X AMPure XP beads (Fisher Scientific, A63880), the fragmentation of 100 pg cDNA was performed with EZ Tn5 Transposase (Lucigen, TNP92110). Fragments of cDNA were amplified by KAPA HiFi hotstart readymix (EMSCO/FISHER, KK2601) with the Nextera i7 primer and Nextera i5 primer. The DNA was purified with 0.8X AMPure XP beads and quantified by qPCR with KAPA Library Quantification Kit (Fisher, NC0078468). The DNA from different samples was pooled at equal molar amounts, and the final sequencing library was loaded at concentrations of 2.7 pM and sequenced on a NextSeq 550 (Illumina) for single-end sequencing.