title: GSM8281019: Raji_ribo-_BS; Homo sapiens; RNA-Seq
library_name: GSM8281019
library_construction_protocol: Total RNA was isolated using TRIzol (Invitrogen) and Direct-zol RNA Miniprep Plus (Zymo Research) according to the manufacturer's protocol. Ribo- RNA was isolated using RiboMinus Eukaryote System v2 (Invitrogen) according to the manufacturer's protocol. PolyA+ RNA was isolated by two rounds of polyA-tailed selection using Dynabeads mRNA DIRECT Purification Kit (Invitrogen) according to the manufacturer's protocol. To remove genomic DNA contamination, RNA was then treated with Turbo DNase and purified by Zymo-IC Column with RNA binding buffer. Library construction of modified BID-seq was conducted similarly with BACS except for the chemical conversion step. For BS treatment, revised BS and desulphonation reaction conditions of BID-seq were used. Briefly, the 3'-ligated RNA was eluted in 10 μl nuclease-free H2O. An 8.5 μl aliquot was subjected to modified BID-seq library construction, while the remaining 1.5 μl was saved as control sample. The 8.5 μl aliquot was mixed with 45 μl freshly prepared BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, Sigma) and incubated at 70 °C for 3 h. The treated RNA was purified by Zymo-IC Column with RNA binding buffer. In-column desulphonation was performed using RNA desulphonation buffer (Zymo), with the column incubated at room temperature for 75 min.