description: Protocols: Pellets were thawed on ice and resuspended in ice-cold buffer B (25mM Tris-HCl pH 7.5; 140mM NaCl; MgCl2 1.8mM, 0.5mM DTT and 0.01% NP-40) supplemented with protease inhibitors (Roche, Complete EDTA free 11873580001). Cells were broken by vortexing with 0.5 mm diameter acid-washed glass beads (Sigma, G8772) 5 times for 80 seconds, alternating with one minute incubations on ice to avoid overheating of the samples. Lysates were clarified by centrifugation at 20000g for 20 minutes, and the supernatants were transferred to new tubes. Protein concentration was quantified by colorimetric assay (Biorad, 5000006), and the samples were stored at -80C until further use. For 2C extraction, the whole protocol is conducted at room temperature and typically, the equivalent of 1 to 2 mg of protein from cell lysates were used per 2C extraction. One volume of lysate was combined with four volumes of lysis buffer. An equal volume of ethanol was added, the sample was mixed, applied to a Zymo-Spin V-E (Zymo Research, C1024) silica column by vacuum, and the flowthrough was discarded. The column was transferred to a collection tube and all subsequent steps were performed by centrifugation at 16000g in a table top microcentrifuge. Samples were consecutively treated with 400 microl of RNA prewash buffer, and twice with RNA wash buffer. The column was then placed on top of a new microcentrifuge collection tube, and purified RNA and RNPs were eluted with 300 microl of water by centrifugation. The RNA concentration was measured using Nanodrop 1000 (Thermo Fisher), and RNA integrity was assessed with Bioanalyzer RNA 6000 nanochips (Agilent, 5067-1511). Pab1-TAP, Tdh3-Protein A and a WT untagged strain were grown in YPD, UV crosslinked and lysed as described above. A first round of 2C was performed from 1mg of protein lysate and 100ug of the obtained 2C-RNA were DNase I treated for 30 minutes at 37C. After DNaseI treatment, RNA samples were diluted with RNA fragmentation buffer to a final concentration of 20mM TrisHCl pH7.5, 1% SDS and 30mM MgCl2. RNA was fragmented by incubating the samples 15 minutes at 95C. Fragmentation was stopped by adding EDTA to a final concentration of 30mM and quickly cooling down the samples on ice and later kept at room temperature. Samples were brought to 2ml with buffer B (25mM TrisHCl 7.5mM; 140mM NaCl, 1.8mM MgCl2; 0.5mM DTT and 0.1% NP-40) and 50 and 100ul were saved for IP validation and input sequencing respectively. To the remaining volume, 100ul of pre-washed Dynabeads Pan mouse IgG (11041, Thermo) were added per sample and were incubated at 4C for 2 hours with gentle rotation. Samples were washed once with buffer B, three times with wash buffer (25mM TrisHCl 7.5mM; 1M NaCl, 1.8mM MgCl2; 0.5mM DTT and 0.1% NP-40) and one time with buffer B. After the washing steps the beads were magnetically pelleted, the supernatant was discarded and the beads were resuspended in 20mM Tris pH7.5. While on the beads, samples were end repaired by the T4 PNK enzyme (NEB, catalogue) following manufacturer instructions. After end repairing, beads were resuspended in 50ul of proteinase buffer plus 5ul of proteinase K (3115828001, Roche) and the samples were incubated for 1h at 37C with gentle rotation. The RNA from the IPs was finally purified by adding to the beads 200ul of RNA lysis buffer. Beads were magnetically pelleted and the supernatants were transferred to new tubes. 250ul of ethanol and 25ul of Magbeads (D4100, Zymo Research) were added per sample. Samples were mixed and incubated for 15 minutes at room temperature with gentle rotation. Beads were magnetically pelleted and washed sequentially with MagBead DNA/RNA Wash1 buffer (R2130-1, Zymo Research) and MagBead DNA/RNA Wash 2 buffer (R2130-2, Zymo Research). After magnetically pelleted, beads were resuspended in 30ul of H2O. Samples were incubated at 37C for 15 minutes and after magnetically pelleting the beads, the eluted RNA was finally transferred to a new tube. The saved 100ul of fragmented 2C-RNA for the inputs were processed in parallel during the 2h incubation period of the immunoprecipitation. Samples were processed for a second 2C extraction using a Zymo-Spin IC micro column and 3ug the resulting RNA were end repaired, in a final volume of 50ul, by the T4 PNK enzyme (NEB, catalogue) following manufacturer instructions. To eliminate the T4 PNK enzyme and buffers, the RNA was later purified by adding 200ul of RNA lysis buffer, after mixing and addition of 250ul of Ethanol, 25 ul of Magbeads were added to each samples. Samples were incubated for 15 minutes at room temperature and gentle rotation. Samples were washed with MagBead DNA/RNA wash buffer 1 and 2 as previously described and RNA was finally eluted in 30ul of H2O. After RNA purification, 1ug of input RNA was subjected to rRNA depletion with the Ribo-Zero Glod Yeast kit (MRZY1324, Illumina) following manufacturer instructions. RNA purified from the IPs and ribodepleted RNA from the input samples were processed for library preparation with the Nextflex Small RNA kit v3 (NOVA-5132-06, PerkinElmer), following manufacturer instructions. Libraries from three biological replicates of inputs and IPs were pooled together and sequenced on a NextSeq500 (Illumina) instrument on a 80 pair end run.
attributes.tag: ENA first public
attributes.value: 2021-05-28
attributes.tag: ENA last update
attributes.value: 2021-05-28
attributes.tag: External Id
attributes.value: SAMEA6979324
attributes.tag: INSDC center alias
attributes.value: EMBL
attributes.tag: INSDC center name
attributes.value: European Molecular Biology Laboratory
attributes.tag: INSDC first public
attributes.value: 2021-05-28T00:10:32Z
attributes.tag: INSDC last update
attributes.value: 2021-05-28T00:10:32Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9263:T3_ProtA_IP_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: baker's yeast
attributes.tag: genotype
attributes.value: Tdh3-protein A
attributes.tag: sample name
attributes.value: E-MTAB-9263:T3_ProtA_IP_1