GSM5912291: Input_DIMT1.rep2; Homo sapiens; OTHER
The cells were harvested by cell lifter after being washed with ice-cold PBS supplemented with CHX (100 μg/ml). The cell pellets were resuspended in lysis buffer (10 mM Tris-HCl at pH 7.4, 150 mM Kcl, 5 mM MgCl2, 100 mg/ml CHX, 0.5% Triton X-100, Protease Inhibitor Cocktail, Rnase inhibitor, and 100 μg/ml CHX) and kept on ice for 15 min. The cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C to remove the cell debris. 100 μl of the cell lysis was aliquoted and 10 μl of 10% SDS was added to it. The Zymo Research RNA Clean &  Concentrator-25 kit was used to purify the total RNA. 200 μl of the cell lysis was aliquoted and 1 U/μl Rnase I was added to it and  kept  at RT for 30 min. Further  Rnase  activity was stopped by addition of SUPERase:In (Ambion). A 10 - 50% w/v sucrose gradient was prepared in the lysis buffer without Triton X-100 by the Gradient Master (BioCamp). The digested cell lysis was layered on top of the gradient and centrifuged at 35,000 rpm for 2.5 hrs at 4°C in a SW-40Ti (Beckman) rotor using an Optima XE-100 Ultracentrifuge (Beckman). Then, the gradient was fractionated into 1 mL by a Biocomp piston fractionator with a TRIAX flow cell for continuous UV detection, and the monosome fractions were pooled. The Zymo Research RNA Clean &  Concentrator-25 kit was used to purify the RPF subsequencially. Obtained RNA was depleted of rRNA with Ribo-minus kit (Invitrogen). For RPF, the RNA was further separated in15% TBE-Urea gel and stained with SYBR Gold. Gel slices containing nucleic acid 27-30 nt were excised and purifed using Zymo small RNA gel purication kit. Libraries were constructed according to NEBNext Small RNA Library Prep kit (NEB) protocol. The concentrations for all the libraries were determined by KAPA Library Quantification Kit (KAPA Biosystems) following the manufacturer's protocol, and subjected to Next-generation high-throughput sequewncing using an illumina NextSeq 550 with a single-end 75-bp read length. Ribo-seq