GSM9678655: Eμ-Myc/+;Prps2^null RNA biol rep 2; Mus musculus; RNA-Seq
B lymphocytes were isolated from lymph nodes suspensions using the EasySep™ Mouse B Cell Isolation Kit (Catalog #19854), respectively, according to the manufacturer's instructions A small portion of the samples were used to prepare Ribo-seq libraries, using the published protocol with some alterations described below. Lymphoma cells were lysed in 20mM Tris pH 7.5, 150 mM NaCl, 5mM MgCl2, 1mM DTT, 100 μg/mL CHX, 1% Triton and 25U/ml Turbo DNase I on ice for 30min by vortexing three times. Lysates were spun down at 10,000g for 10 min, and the RNA concentration was measured by Qubit RNA HS Assay kit (Invitrogen). 30 μg and 1μg of RNA containing lysates were used to process Ribo-seq and RNAseq libraries, respectively. 30 μg RNA containing lysate was digested with 1.5 µl RNase I (10 U/µl by the Epicentre definition) and incubate for 45 minutes at room temperature with gentle agitation. RNase digestion was inhibited by adding 10ul SuperaseIN RNase Inhibitor and immediately putting the samples on ice. Ribosomes with footprints were isolated using MicroSpin S-400 column. RNAs were isolated by mixing with TRizol-LS and precipitated by isopropanol and glycogen. Next, purified RNA samples were run on a 15% TBE-urea gel, stained by SyberGold, and the bands above 24nt and below 34 nt were extracted from the gel. Dephosphorylation and linker ligation of the footprint fragments followed by reverse transcription, circularization of cDNA, and the library construction were prepared. For the RNAseq libraries, RNA was isolated by TRizol from the lymphoma lysates. Only 10ng of total RNA was used following the Takara Smarter Stranded Total RNA-Seq kit protocol, and both libraries were submitted to the UCSF CAT facility.