Name: CTL_RPF_1_s
Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: MNase
Layout: SINGLE
Construction protocol: Meis1/Hoxa9-transduced foetal liver c-Kit+ cells were collected after three rounds of serial re-plating in methylcellulose. Cells were lysed with CaCl2 4 mM, MgCl 10 mM, K-HEPES pH 7.2 25 mM, KOAc 200 mM and NP-40 1%. Total RNA was extracted using QIAzol Lysis Reagent (QIAGEN). The lysate was cleared from cell debris, diluted 1:1 in water, and digested with MNase 10 μg/ml for 30 minutes at 37°C. Digested RNA was extracted with QIAzol and later treated with PNK (NEB) for 30 minutes at 37°C. To isolate ribosome-protected mRNA fragments (RPFs), the PNK-treated RNA was resolved on a 15% Novex TBE-Urea Gel (EC6885BOX), and RPFs 25 to 40 nucleotides long were excised and purified. Libraries were then prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina following manufacturer's instructions.