Instrument: NextSeq 550
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For cell harvest, growth medium was poured off and cells were quickly washed with ice-cold washing solution (1x PBS 10 mM MgCl2 800 µM Cycloheximide). Washing solution was immediately poured off and freshly prepared crosslinking solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 0.025% PFA, 0.5 mM DSP) was added to the cells. Cells were incubated with crosslinking solution for 15 minutes at room temperature while slowly rocking. Crosslinking solution was then poured off and remaining crosslinker was inactivated for 5 minutes with ice-cold quenching solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 300 mM Glycine). Quenching solution was poured off and 150 µl of lysis buffer (0,25 M HEPES pH 7.5, 50 mM MgCl2, 1 M KCl, 5% NP40, 1000 μM CHX) was added to each 15 cm dish, resulting in 750µL of lysate. Lysis was carried out at 4°C. After RNA extraction from total and IP-purified fractions, RNA quality and integrity were determined on an Agilent Bioanalyzer using the total RNA Nano 6000 Chip. For size selection, RNA was run on 15% Urea-Polyacrylamide gels (Invitrogen) and fragments of size 20-60 nt (80S libraries) and 20-80 nt (40S libraries) were excised using the Agilent small RNA ladder as a reference. RNA was extracted from the gel pieces by smashing the gels into small pieces with gel smasher tubes and extracting the RNA in 0.5 ml of 10 mM Tris pH 7 at 70°C for 10 minutes. Gel pieces were removed and RNA was precipitated using Isopropanol. Footprints were then dephosphorylated using T4 PNK (NEB) for 2 hours at 37°C in PNK buffer without ATP. Footprints were then again precipitated and purified using isopropanol. For 40S footprints, contaminating 18S rRNA fragments were depleted as follows. Prevalent 18S rRNA fragments from the first round of 40S footprinting were used to design complementary Biotin-TEG-DNA oligonucleotides (sequences listed in Supplemental Table 4, ordered from Sigma-Aldrich). 100ng of RNA footprints were then hybridized to a mixture (proportional to occurrence of the fragment, listed in Suppl. Table 4) of these DNA oligos (in 40x molar excess) in (0.5M NaCl, 20mM Tris pH7.0, 1mM EDTA, 0.05% Tween20) by denaturing for 90 sec at 95C and then annealing by reducing the temperature by -0.1C/sec down to 37°C, then incubating 15min at 37°C. Hybridized species were pulled out using Streptavidin magnetic beads (NEB) by incubating at room temperature for 15 minutes, and the remaining RNA was purified by isopropanol precipitation. Footprints were then assayed using an Agilent Bioanalyzer small RNA chip and Qubit smRNA kit. 25 ng or less of footprint RNA was used as input for library preparation with SMARTer smRNA-SeqKit for Illumina from Takara / Clontech Laboratories according to the manufacturer's instructions. Deep-sequencing libraries were sequenced on the Illumina Next-Seq 550 system.