Illumina HiSeq 2500 sequencing; Ribosome profiling of CLL patient samples
Ribosome profiling of CLL patient samples
Cycloheximide (100 µg/ml) was added to the wells for 5 min at 37deg to stop translation and, working at 4deg C, CLL cells were removed from the plates by pipetting and transferred to 15 ml Falcon tubes on ice prior to centrifugation at 300g, 4deg C for 3 minutes.   The pellet was resuspended in 5 ml of ice cold PBS with cycloheximide (100 µg/ml) and centrifuged again at 300g, 4deg C for 3 minutes.   Following the addition of 0.5 ml lysis buffer (3M NaCl, 150mM MgCl2, 150mM Tris-HCl (pH7.5), 20mM DTT, cycloheximide (1mg/ml), 1% Triton X-100 and RNasin (12.4µl/ml), lysates were centrifuged at 3000 rpm, 4deg C for 5 minutes before being snap frozen in liquid nitrogen and stored at -80deg C. Fibroblasts expressing CD40L were irradiated (35 Gy for 15 minutes) and placed on a 6-well plate so as to produce an ~80% confluent culture.   1 day following irradiation CLL cells at 3x106 cell/ml were added to this feeder layer with IL-4 (10 ng/ml).   CLL cells were cultured for 72 hours before harvesting. RNase I (0.5 U/µl) was added to lysates for 30 minutes at room temperature with gentle rotation.   Lysates were then layered on sucrose gradients and centrifuged at 38000 rpm, 4deg C for 2 hours in a Sorvall centrifuge with settings acceleration 9 and deceleration 6 with a SW-41 Ti rotor.   Samples were fractionated using FoxyR1 Fractionation System and RPF UV traces were compared (plus/minus RNAse I) to determine those fractions (typically 9 to 11) that contained ribosome protected fragments (RPF).   Trizol LS (1.4 ml) was added to the fractions, mixed, and allowed to stand for 5 minutes at room temperature to dissociate the nucleoprotein complexes.   Bromochloropropane (BCP) (200 µl) was added and samples were shaken for 15 seconds to mix and allowed to settle for 5 to 10 minutes prior to centrifugation in a refrigerated centrifuge for 15 minutes at 4deg C.   The aqueous layer was carefully removed and isopropanol (1 ml) and GlycoBlue (1 µl) were added.   Following incubation at room temperature for 10 minutes the lysates were centrifuged at 13000 rpm in a refrigerated microfuge at 4deg C.   The supernatant was removed and the pellet washed with 75% ethanol followed by a further centrifugation for 10 minutes at 13000 rpm in a refrigerated microfuge at 4deg C.   The pellet was washed, air dried for 5 to 10 minutes and resuspended in 10 µl ultrapure water and stored at -80deg C.   RPFs were size selected on a PRE-cast 15% TBE-urea gel.   RNA (10 µl) was mixed with 2x sample buffer (7M Urea, 12% Ficoll, 1xTBE, 0.03% bromophenol blue, 0.03% xylene cyanol) (10 µl).   28-mer and 34-mer size marker controls (500 nM) were similarly mixed with 2x sample buffer and both samples and controls were heated at 80deg C for 2 minutes and then snap cooled on ice prior to loading and running at 180 V for about 75 minutes.   RNA was visualised and the RPFs, defined by the 28-mer and 34-mers, were excised prior to elution by shredding, addition of 300µl pure water and rotation overnight at 4deg C;  followed by precipitation with ethanol and sodium acetate.   The pellet obtained by centrifugation in a refrigerated microfuge at 13000 rpm for 30 minutes at 4deg C was washed with 75% ethanol, and centrifuged again for 10 minutes at 4deg C.   The supernatant was removed and the pellet allowed to air dry following which it was resuspended in ultrapure water (10 µl) and stored at -80deg C. This RPF sample was end-repaired using PNK enzyme, and then the library was prepared using the TruSeq Small RNA library prep kit (Illumina).
Experimental Factor: individual
ID30