GSM8586644: Alr1700-3xFLAG, rep2; Nostoc sp. PCC 7120 = FACHB-418; RIP-Seq
Proteins from the eluted RNA-protein complexes were digested in 400 µL proteinase K buffer (50 mM Tris-HCL, pH 6, 5 mM EDTA and 0.5%SDS) with 400 µg of proteinase K for 30 min at 37 °C. Another 400 µL of proteinase K buffer + 9M urea was added for an additional incubation of 30 min at 37 °C. RNA fragments were isolated using  phenol-chloroform and chloroform extractions. RNA was later treated with 2U of TURBO DNAse (Life Technologies) to eliminate DNA traces, followed by RNA cleanup using RNA Clean & Concentrator columns (Zymo Research). Synthesis of cDNA was facilitated by SMARTScribe reverse transcriptase (TaKaRa), which adds the loop adapter at the 3'-end in the first-strand synthesis step and the SMART oligo at the 5'-end in the subsequent template switching and extension step. Annealing of the loop adapter to RNA was achieved by incubating both at 72°C for 3 min and 42°C for 2 min. All components were added to this mixture according to the manufacturer's instructions, including the SMART oligo, 100 U SMARTScribe reverse transcriptase (TaKaRa), and 40 U of Ribolock RNase inhibitor (Life Technologies), and the reaction was incubated at 42°C for 90 min and 70°C for 10 min, followed by cooling to 12°C. After cDNA synthesis, the samples were excised from 3% Nusieve agarose gels and purified with the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel), using NTC buffer to solubilize the gel slices. The cDNA was amplified in 22 PCR cycles with Phusion high-fidelity DNA polymerase (New England BioLabs) using Illumina 8-nt index primers for both sides, and PCR samples were purified using the Agencourt AMPure XP system (Beckman Coulter) according to the manufacturer's instructions. All individual amplified cDNA libraries were pooled in equimolar ratios and size selected between 100 - 320 bp using the Pippin Prep system (Sage System) according to the manufacturer's instructions and subsequently purified using the Agencourt AMPure XP system (Beckman Coulter).