title: GSM4490919: Trx-iCLIP (neg.ctr.) rep1; Saccharolobus solfataricus; OTHER
library_construction_protocol: For cell lysis, 0.5 g of cells were thawed on ice, resuspended in lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% Glycerol, 0.1% Nonidet P40, 1 mM PMSF) and lysed by sonication (five 30 sec cycles, 70%). Cells debris was removed by centrifugation, and supernatant was DNase- and RNase-treated. 10x RQ1 DNase buffer was added to 1x final concentration, and 1:500 vol. Turbo-DNase (Ambion), 1:1,000 vol. RNaseOUT (Thermo Fisher Scientific) and different dilutions of RNase I (Ambion), in RQ1-buffer (high RNase: 1:1,000 vol. and low RNase 1:10,000 vol.). Extracts were incubated for 6 min at 37°C in a shaking water bath. All following steps were performed on ice / at 4°C. Immunoprecipitation was performed as described in Walter et al. (2006) (PMID: 17078816): 4.5 ml of lysate was incubated with Protein-A-Sepharose beads coupled to 100 μl of polyclonal antibody raised against His-tagged Rrp41- or Rrp4-: against His-tagged Rrp41 and Rrp4 (Witharana et al., 2012) (PMID: 22503705), or polyclonal antibody raised against Thioredoxin (Trx) from the alphaproteobacterium R. sphaeroides (Li et al., 2003) (PMID: 12624204)  as a negative control for 2 h. Beads were washed ten times with high salt wash buffer (10 mM Tris pH 7.5, 1 M NaCl, 5 mM MgCl2, 10% Glycerol, 0.1% Nonidet P40, 1 mM PMSF). To remove excess of salt, beads were then washed two times with PNK-buffer (70 mM Tris pH 7.5, 10 mM MgCl2, 0.05% NP-40). All following steps were performed as described in König et al. (2010). In brief: immunoprecipitated crosslinked RNA-protein complexes were subjected to several enzymatic reactions on-bead. Subsequent de-phosphorylation of RNA 3'-ends by phosphatase-treatment, a 3'-RNA linker ligation and ³²P-5'-end labelling of the RNA using T4 polynucleotide kinase and gamma-[³²P]-ATP were performed. Complexes were resolved on a denaturing neutral-pH SDS-polyacrylamide gel electrophoresis (NuPAGE, Invitrogen), and transferred to a nitrocellulose membrane Protein-RNA-complexes were visualized by autoradiography on an x-ray film at -80°C. Complexes of adequate size were excised from the membrane and RNA was isolated by proteinase K treatment. iCLIP library preparation was performed as described in König et al. (2010), and sequencing on an Illumina MiSeq system, 75 bp single-read.  Following oligonucleotides were used:  3'-RNA linker (L31): P-UGAGAUCGGAAGAGCGGUUCAG-Puromycin  Reverse transcription primers (containing random and experimental barcode,):  R1clip P-NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC  R6clip P-NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC  R9clip P-NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC  R10clip P-NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC  R13clip P-NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC  R14clip P-NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC
attributes.tag: GEO Accession
attributes.value: GSM4490919