title: GSM8651109: MOLM13-RNAseq-FP54-rp1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated using Trizol method and precipitated using ethanol. RNAseq and m6A seq: NB4 or MOLM-13 cells were incubated with DMSO or FP-54 (2 μM) for 48 h at 37 °C and 5% CO2, cells were collected by centrifugation, washed with PBS, and suspended in TRIzol reagent (Invitrogen). Total RNAs were extracted following the manufacturer's protocol through ethanol precipitation. mRNA enrichment: mRNA was purified with two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion) following the manufacturer's protocol. RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific). 1 μg mRNA for each sample were fragmented and enriched by m6A antibody (Millipore). Fragmented mRNA (as “input”) and immunoprecipitated RNA (as “IP”) were subjected to RNA library construction following manufacturer's instructions of SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara). polysome-seq: 20 million MOLM-13 cells were incubated with FP-54 (2 μM) for 48 h, Cycloheximide (CHX) was added to the media at 100 mg/ml and incubated at 37°C for 7 min. Cells were collected by centrifugation, washed with ice-cold PBS with CHX (100 mg/ml), the cell pellet was suspended in lysis buffer which was formulated as 20 mM HEPES, pH 7.6, 100 mM KCl, 5 mM MgCl2, 100 μg/ml CHX, 1% Triton-X-100, freshly add 1:100 protease inhibitor (Roche), 40 U/ml SUPERasin (Ambion). The lysate was loaded onto the sucrose gradient and centrifuged at 4°C at 28,000 rpm (Beckman, rotor SW28) for 3 h. The samples were then fractioned and the A260 absorbance of each fraction was analyzed. Total RNAs of polysome fractions were suspended in TRIzol LS (Invitrogen), and RNA were extracted by RNA Clean & Concentrator (Zymo), mRNAs were then purified with two rounds of polyA+ purification with Dynabeads mRNA DIRECT kit (Ambion), and the libraries were constructed using SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara).