Ss 34fixed angle rotor

Cells were grown in LB medium supplemented with 5 μg/ml of
chloramphenicol and, when indicated, with 20 μM bortezomib for 8 h,
at 37°C until OD₆₀₀ = 1.5-2.0. Cells were disrupted with a
Microfluidizer Emulsi Flex C5 (Avestin) in 50 mM Tris-HCl pH 7.4, 100 mM
NaCl, 5 mM MgCl₂, 5% Glycerol, 0.1% NP-40, 1 mM DTT and protease
inhibitor mixture. After clearing by centrifugation at 17,000 rpm in SS-34
fixed-angle rotor (Thermo) for 22 min at 4°C, lysates were incubated
for 1 h at 4°C with GFP-Trap agarose beads. Beads were washed 3 times
with lysis buffer followed by elution of bound proteins with 0.2 M Glycine
pH 2.5 and neutralization with 1 M Tris pH 10.5. Proteins in the eluate were
precipitated with 10% TCA (final concentration), resuspended in sample
buffer, and separated on 4–12% NuPAGE Bis-Tris gel (Invitrogen). Gels
were stained by Colloidal Blue Staining Kit (Invitrogen) and bands were cut
for MS analysis.

Cells were grown and disrupted as above, except that the lysis
buffer contained ribonuclease inhibitor (Sigma). After clearing by
centrifugation at 17,000 rpm in SS-34 fixed-angle rotor (Thermo) for 22 min
at 4°C, lysates were incubated for 2 h at 4°C with anti-FLAG
M2 Affinity Gel. Beads were washed 3 times with 50 ml of lysis buffer
followed by elution of bound material with 3X FLAG Peptide. Total RNA was
isolated from elution as well as input samples with TRIzol reagent, per the
manufacturer’s manual. 300 ng of total RNA from each sample were run
on the Fragment Analyzer™ Automated CE System using DNF-471 Standard
Sensitivity RNA Analysis Kit (15nt) following the manufacturer’s
instructions.
For small RNA sequencing, 100 ng of RNA purified as above were
processed with the Small RNA-Seq Library Prep Kit (Lexogen) following the
manufacturer’s instructions. Library quality and quantity were
assessed in a Fragment Analyzer using the DNF-473 Standard Sensitivity NGS
Fragment Analysis Kit (1 bp - 6000 bp), as well as with a Qubit dsDNA BR
Assay Kit (Q32854, Thermo Fisher scientific). Libraries were multiplexed and
sequenced on an Illumina HiSeq200 platform to obtain 50-nt single-end
reads.