title: GSM4084997: designed_library_25-100_length_utrs; Homo sapiens; RNA-Seq
library_construction_protocol: Salt solution (10x): 100 mM NaCl, 100 mM MgCl2,100 mM Tris-HCl pH 7.5, and RNase-free water (Zuccotti P, Modelska A 2016). Wash buffer: 100 µg/ml cycloheximide in RNase-free DPBS (10 ml per plate). Lysis buffer: 1x salt solution, 1% of 20% Triton X-100, 1 mM DTT, 0.2 U/µl SUPERase-In (Thermo Fisher Scientific), 100 µg/ml cycloheximide. Wash buffer and lysis buffer were chilled throughout the protocol. After 12 hours of growth at 37 °C, cells were placed on ice and media was aspirated. Translating ribosomes were halted by adding 5 ml of wash buffer and were then placed at 37 °C for 5 minutes followed by aspiration on ice. Washed by adding 5 ml wash buffer and aspirating thoroughly. Added 300 µl of ice-cold lysis buffer, scraped cells, broke up cell clumps by pipetting ~5 times, and then placed suspended cells into a pre-chilled microcentrifuge tube. Let sit for 10 minutes on ice and then triturated by passing through a 25-G needle 10 times [1]. Spun at 16,000 x g for 5 minutes to pellet cell debris and nuclei. Saved supernatant and added 1.5 µl of 1 U/µl DNase I (final concentration of 0.005 U/µl) and placed on ice for 30 minutes. Either stored at -80 °C or proceeded directly to polysome profiling. For both the eGFP and mCherry library, randomized 50-mer oligos were Gibson assembled to serve as 5' UTRs for each CDS. To create the designed library, oligos were made via oligo synthesis array (CustomArray Inc.) and cloned into the eGFP vector.For all libraries, 25 nucleotides of defined sequence preceed the inserted 5' UTRs.