GSM4008949: eCLIP Thermus thermophilus, normal-growth conditions size-matched input, replicate 2; Thermus thermophilus HB27; RIP-Seq
The isolation of RNA-protein complexes, trimming of the RNA, and preparation of the cDNA library was performed as described (Van Nostrand et al. 2016). Briefly, cells were thawed in lysis buffer (50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1% (w/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium desoxycholate, and Protein Inhibitor Cocktail III (1:200, EMD Millipore) and disrupted by sonification. The crude extract was treated with DNase and RNase to remove DNA and to trim the protein-bound RNA. Hera-RNA complexes were immunoprecipitated using an anti-Hera antibody from rabbit (Eurogentec), and isolated using sheep anti-rabbit Dynabeads. Subsequent steps were performed with RNA bound to the beads. After dephosphorylation with FastAP (ThermoFisher) and T4 PNK (NEB), a barcoded RNA adapter was ligated to the 3′ end (T4 RNA Ligase, NEB). RNA-protein complexes were separated by SDS-PAGE and transferred to nitrocellulose membranes. The region from 55-130 kDa (75 kDa above the molecular mass of Hera) was excised, the membrane was suspended in buffer and treated with proteinase K (NEB). RNA was isolated by phenyl/chloroform extraction, purified on spin columns (Zymo Research), and reverse-transcribed with AffinityScript (Agilent). Excess oligonucleotides were removed by treatment with ExoSAP-IT (Affymetrix). The second DNA adapter (containing a random-mer of 5 (N5) or 10 (N10) random bases at the 5′ end) was ligated to the 3'-end of the cDNA (T4 RNA Ligase, NEB). After cleanup on Dynabeads MyOne Silane (ThermoFisher), the required number of PCR cycles was determined from qPCR experiments, the DNA was amplified by PCR, purified with the PippinPrep system (Sage Science), and sequenced on the Illumina HiSeq.