title: GSM8421669: Transcriptome BCAA vehicle AML cells biol rep4; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized at the point of maximum response of chemotherapy and femurs, tibias, pelvis and humeri were isolated and cleaned to remove muscle. Bones were then crushed using a pre-cooled mortar and pestle in ice-cold flow buffer (PBS containing 2% FBS), and the obtained BM cell suspension was passed through a 70 μm cell strainer. Red blood cells were removed by incubating cells for 5 minutes in ACK lysing buffer (Gibco) on ice, the remaining BM cells were washed and resuspended in ice-cold flow buffer. Viable cells (AML cells GFP+) were sorted on a BD FACSAria II (BD Biosciences) with the sample collector cooled to 4°C. After centrifugation, samples were lysed in 20 μl ice-cold lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX, 1% Triton X-100; 25 U/ml Turbo DNase) for 10 min on ice. For mRNA 2 μl of the lysate was subjected to the Smart-seq2 protocol. Following addition to 300 μl lysis buffer to the lysate, it was clarified for 10 min at 20,000g at 4°C, then treated with RNase 1 Ambion, AM2295, 1 μl for up to 50k cells, 2 μl for up to 200k cells) and incubated at room temperature for 45 min with gentle mixing. The digested extracts were stopped by the addition of 10 μl SUPERaseIn and were overlaid onto a 700 μl sucrose cushion of 1 M sucrose in 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml CHX and 20 U/ml SUPERaseIn for 4 h at 260,000g, 4°C in MLA-150 rotor in Beckman Optima MAX-XP ultracentrifuge. The supernatant was removed, and the pellet was resuspended in 50 μl pellet buffer (10 mM Tris pH 7.5 and 1% SDS). RPFs were extracted from pellet buffer by using 1 ml Trizol and 200 μl chloroform. The aqueous phase was precipitated by adding 750 μl isopropanol and 20 μg glycogen. Precipitation was carried out at −20 °C overnight, and RPFs were then pelleted by centrifugation for 40 min at 12,000 g, 4 °C. RPF was resuspended in 6 μl nuclease-free water and mixed with 6 μl Gel Loading Buffer II. The 15% (wt/vol) polyacrylamide TBE-urea gel was pre-run for 30 min at 200 V. The RNA samples were denatured for 90 s at 80°C. RPFs were separated by electrophoresis for 65 min at 200 V. To minimize contamination, each sample was run in a separate gel and the electrophoresis was run without RNA or DNA markers. The region from 26 to 34 nt for RPFs was excised according to the light-blue loading dye and further determined on the basis of the relative position of RNA oligo markers to the loading dye on a separate and parallel gel. Extract RNA from the polyacrylamide gel slices by adding 400 μl RNA extraction buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA and 0.25% SDS), snap-frozen at −80°C for 20 min and thawed at 37°C for two rounds and then kept overnight at room temperature with gentle mixing. The liquid was transferred to a clean tube, and 500 μl isopropanol and 20 μg glycogen were added and precipitated overnight. The RPF sample was pelleted by centrifugation for 40 min at 12,000 g, 4°C and resuspended in 6 μl nuclease-free water. The sample was then subjected to sequencing library preparation by D-Plex Small RNA-seq Kit and DNA library was purified using AMPure XP. Barcoded libraries were pooled and sequenced on the Illumina platform with 2x 100bp reads. For mRNA 2 μl of the lysate (10%) was subjected to the Smart-seq2 protocol. The Ribo-lite/RPF sample was then subjected to sequencing library preparation by D-Plex Small RNA-seq Kit and DNA library was purified using AMPure XP.