GSM5234270: APEX-seq APEX2EGFP_wArs_rep1; Homo sapiens; RNA-Seq
After the cellular labeling, total RNA was purified using TRIzol per the manufacturer's instruction. To enrich biotinylated RNAs, Pierce streptavidin magnetic beads (Thermo Fisher) were used (20 μL beads per 50 μg RNA). The beads were washed 3 times with 500 μL B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, and 0.1% TWEEN 20), followed by 2 times of 500 μL solution A (0.1 M NaOH and 50 mM NaCl), and 1 time of 500 μL solution B (100 mM NaCl). The beads were then suspended in 125 μL solution B. 50 μg RNA diluted in 125 μL water was mixed with the beads and incubated at 4 °C for 2 hrs with rotation. Then, the beads were placed on a magnetic stand to remove the solution and washed 3 times with 500 μL B&W buffer. Finally, the beads were resuspended in 54 μL water. To release the biotinylated RNA, 33 μL proteinase buffer (3 x PBS, 6% N-Lauryl sarcosine sodium solution (Sigma-Aldrich), 30 μM EDTA, and 15 μM DTT) was added to the beads with 10 μL proteinase K (Thermo Fisher) and 5 μL RNase inhibitor. The beads were then incubated at 42 °C for 1 hr and 55 °C for 1 hr on a shaker at 600 rpm. The RNA was then purified using the RNA clean and concentrator 5 kit (Zymo Research) following the manufacturer's instruction. The resulted RNA was then used for RNA-seq library construction. Libraries were constructed according to TruSeq Stranded mRNA Kit (Illumina) protocol. The concentrations for all the libraries were determined by KAPA Library Quantification Kit (KAPA Biosystems) following the manufacturer's protocol, and subjected to Next-generation high-throughput sequewncing using an illumina NextSeq 550 with a single-end 75-bp read length.