title: GSM8148830: Ngn2 iNeurons ribosomeFootprinting 7Dup isoDup (242K) rep2; Homo sapiens; OTHER
library_construction_protocol: iNeurons were treated with cycloheximide at 0.1mg/mL for 5 min and lysed according to the manufacturer's instructions, then cell lysates were digested with TruSeq Ribo Profile Nuclease. The Ribosome Protected fragments were first purified using MicroSpin S-400 columns (GE) and then size-selected (28-30 nt) from 15% polyacrylamide/7-8 M urea/TBE gel. rRNA was removed using the Ribo-Zero Gold Kit (Illumina). After 3' adaptor ligation and reverse transcription of the library, cDNA (70-80 nt) was purified from 10% polyacrylamide/7-8 M urea/TBE gel. After cDNA circularization and limited amplification (9 cycles), the RPF libraries were purified using an 8% native polyacrylamide gel.