title: GSM4832656: CD42 positive megakaryocytes replicate 2; Homo sapiens; OTHER
library_construction_protocol: Cell lysis and RNA isolation for ribosomal footprinting was performed according to Ignolia et al. with some adaptations. Briefly, CD42- and CD42+ selected megakaryocytes were pelleted. Cell pellets were dissolved in 150uL ice cold lysis buffer (20mM Tris pH 7.4, 250mM NaCl2, 5mM MgCl2, 0.5% Triton X-100, 1mM Dithiotreitol (DTT), 0.024U/uL Turbo DNAse (Ambion), 0.01U/uL RNAseOUT (Invitrogen) and 100g/mL Cycloheximide). The lysate was immediately used for ribosomal footprint preparation. Samples were kept on ice for 10 minutes and regularly vortexed to fully lyse the outer membranes and release the ribosomes. Lysates were spun at 20.000g for 10min at 4ºC and supernatant was harvested to remove the remaining pelleted cell debris and nuclei. Lysates were treated with 2uL of RNAse 1 (Ambion) and incubated at 37ºC for 45 minutes. The reaction was stopped by the addition of 5uL SUPERAseIN RNAse inhibitor. Lysates were loaded on a 1M 0.150mL Sucrose cushion in 8x34 mm polycarbonate thick wall tubes and centrifuged at 120.000g for 30 minutes in a TLA-120.1 rotor in a pre-cooled 4ºC table top ultracentrifuge. The pellet was resuspended using 100uL resuspension buffer (10mM Tris pH 7.0, 1% SDS and 0.01U/uL proteinase K (NEB)) for 30 minutes at 37ºC. Footprints were subsequently extracted with miRNAeasy kit according to manufacturer's guidelines for small RNA. rRNA contamination was removed using the Ribo-Zero gold rRNA removal kit (epicenter/Illumina; 20020596). Footprint RNA quality was assessed using the Agilent Bioanalyzer using the RNA chip. Footprints were selected for 26-34nt with custom markers on Elchrom Scientific ORIGINS system and short fragment gels, using gel electro-elution into Spectra/Por 3 3.5KD MWCO dialysis membranes. Libraries were produced using the Art-Seq kit (Epicentre/Illumina) according to the manufacturer's protocol. The quality of libraries was assessed on Agilent Bioanalyzer with high sensitivity DNA chip and quantified using qPCR with the KAPA library quantification kit (KK4824). Libraries were pooled to a 2nM concentration and 50bp single-end reads were sequenced on HiSeq 2000 (Illumina). 50bp single-end reads were sequenced on HiSeq 2000 (Illumina).