GSM5019832: CD34neg_1 [Ribo-seq]; Mus musculus; OTHER
RNA-seq: Sorted cells were frozen in liquid nitrogen and lyszed in 300µl lysis buffer (10mM NaCl, 10mM MgCl2, 10mM Tris-HCl pH7.5, 1% triton X-100, 1% sodium deoxycholate, 1mM DTT, 0.1mg/ml cycloheximide). 1µg of total RNA was extracted using the Direct-zol RNA Microprep Kit (Zymo Research) according to manufacturer's instruction. RNA-seq: Libraries were prepared using the NEBNext® UltraTM RNA Library Preparation Kit for Illumina (NEB) including hybridization-based ribo depletion according to manufacturer's instructions. Library validation and quantification were performed using TapeStation analysis (Agilent). Equimolar amounts of the library were pooled. The pool was quantified using the KAPA Library Quantification Kit (Peqlab) and the ABI Prism® 7900HT Sequence Detection System (Applied Biosystems) and sequenced on an Illumina NovaSeq6000 sequencer. Ribo-Seq: Sorted cells were frozen in liquid nitrogen and lyszed in 300µl lysis buffer (10mM NaCl, 10mM MgCl2, 10mM Tris-HCl pH7.5, 1% triton X-100, 1% sodium deoxycholate, 1mM DTT, 0.1mg/ml cycloheximide). For the isolation of ribosome-protected fragments, the samples were incubated with RNase I and S7. Ribosomal proteins were digested by proteinase K for 1h at 65°C. RNA fragments were purified by acid-phenol:chloroform extraction and precipitated by the addition of cold ethanol. Ribo-Seq: The fragmented RNA pellets were resuspended in nuclease free water and treated with T4 Polynucleotide kinase (NEB) for 90 min at 37°C. Size selection of the fragmented RNA was performed using PAGE. Gel slices in the range of 24 36 nt were excised using X Tracta Tips (Biozym). After purification, RNA fragments were resuspended in nuclease free water. Sequencing libraries were prepared using the Small RNA Seq Library Prep Kit (Lexogen) according to manufacturer's instructions. Amplification was done using 16 PCR cycles. The pool of all libraries was size-selected using the BluePippin device (SAGE-Science) with a 3% DF Marker Q3 Kit (SAGE-Science). The range of 135 180 bp was selected. The pool was quantified using the KAPA Library Quantification Kit (Peqlab) and the ABI Prism® 7900HT Sequence Detection System (Applied Biosystems) and sequenced on an Illumina NovaSeq6000 sequencer.