title: Illumina HiSeq 2500 sequencing; Ribosome profiling of CLL patient samples
design: Ribosome profiling of CLL patient samples
library_construction_protocol: 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. 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.   Total RNA samples were submitted to two rounds of poly-A selection using the Invitrogen RiboMinus Transcriptome Isolation kit, and RiboMinus Concentration Module.   Briefly, total RNA was hybridised to the RiboMinus probe, and the resulting rRNA/probe complex removed using RiboMinus beads.   The remaining sample was mixed with binding buffer and ethanol, and purified using the RiboMinus Concentration Module spin columns.   Columns were washed twice with wash buffer and RNA was eluted in RNase-free water.   This was done twice. This total RNA sample was used to prepare the whole transcriptome (WT) library using the TruSeq Stranded mRNA library prep kit (Illumina).
attributes.tag: Experimental Factor: individual
attributes.value: ID30