title: GSM7371602: Brain Organoid, MTK, Day 60, Riboseq, biol rep 3; Homo sapiens; OTHER
library_construction_protocol: For RNA-seq, total mRNA was extracted from D60 organoids treated with DMSO, MTK or DEAB by using the RNeasy Mini kit. For Ribo-seq, cellular RNA were extracted by mild lysis buffer and Lysates oforganoids were treated with 750 U RNase I for 45 minutes at room temperature. Samples were then transferred to an ultracentrifuge tube on a sucrose cushion (~34%) and centrifuged at 70,000 rpm at 4C for 4 hr. RNA libraries for bulk RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following manufacturer's protocols. For riboseq, Ribosome-protected fragments were purified using miRNeasy RNA isolation kit (QIAGEN, cat. No. 217004). RNA was size selected by running a polyacrylamide gel, and then dephosphorylated, linker ligated, and then subjected to RT-PCR, circularization, rRNA depletion, and 10-12 cycles of PCR. The enzymes used were T4 PNK (NEB, cat. No. M0201S), T4 RNA ligase 2, truncated (NEB, cat. No. M0242S), Universal miRNA Cloning Linker (NEB, cat. No. S1315S), Superscript III (Invitrogen, cat. No 18080-044), Circ Ligase (Epicentre, cat. No. CL4111K), NEBNext® High-Fidelity 2X PCR Master Mix (cat. No. M0541L). Resulting fragments were size selected from an 8% acrylamide non-denaturing gel and purified by incubation overnight with DNA gel extraction buffer (300 mM NaCl, 10 mM Tris (pH 8) and 1 mM EDTA).