GSM5456557: RNA_0h_KCl_rep3; Homo sapiens; RNA-Seq
Ribosome profiling was performed using a protocol modified from McGlincy et al. Frozen brain tissue (~80 mg) was thawed on ice. Each sample was dounced 15x in 400 µL ice cold lysis buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide (Sigma). The lysate was further sheared using a 26-gauge syringe. The lysate was clarified by centrifugation at 20,000 x g for 10 minutes at 4ºC. Supernatant (10 µL) was removed, added to 300 µL Trizol, and frozen at -80ºC for future RNA-seq library preparation. RNA concentration in the remaining supernatant was quantified using RNA Qubit. Lysate (30 µg) was subjected to RNase I digestion (0.5 U RNaseI per µg RNA) at room temperature for 15 minutes with gentle agitation. After RNase digestion, 10 µL SuperasIN (Thermo) was added to each sample, and the samples were transferred to ice. To isolate ribosome protected fragments, the RNase-digested lysate was transferred to Ultra-clear 11x34 mm centrifuge tubes (Beckman Coulter) and underlaid with 0.9 mL sucrose cushion. Samples were centrifuged in a TLS-55 rotor at 51,000 rpm for 2 hours at 4°C. The supernatant was discarded and the pellet was resuspended in 300 µL TRIzol. Ribosome-protected fragments were purified from TRIzol using the Zymo Direct-zol kit. RNA was precipitated by adding 38.5 µL RNase-free water, 1.5 µL Glycoblue, 10 µL 3 M sodium acetate pH 5.5, and 150 µL isopropanol to 100 µL eluted RNA. The mixture was incubated overnight at -20°C. Samples were centrifuged for 30 minutes at 20,000 x g at 4°C. The supernatant was discarded, and the RNA pellet was resuspended in 5 µL 10 mM Tris pH 8. Five µL 2X denaturing sample loading buffer (980 µL formamide, 20 µL 500 mM EDTA, 300 µg bromophenol blue) was added to each sample, then the sample was denatured at 80°C for 90 seconds. Ribosome-protected fragments, along with control oligos66, were run on a 15% polyacrylamide gel at 200 V with 12 µL NEB miRNA marker. The gel was stained with SYBR gold in 1X TBE. Gel fragments between 17 and 34 nucleotides were excised and placed in a microfuge tube with 400 µL gel extraction buffer. Samples were frozen on dry ice for 30 minutes, then thawed overnight with gentle agitation. After overnight gel extraction, 400 µL eluate was transferred to a new microfuge tube. The RNA was precipitated by adding 1.5 µL glycoblue and 500 µL isopropanol. After overnight incubation at -20°C, the sample was centrifuged at 20,000 x g for 30 minutes at 4°C. The supernatant was discarded, and precipitated RNA was resuspended in 4 µL 10mM Tris pH 8. Samples were then dephosphorylated using T4 PNK (4 µL RNA in 10 mM Tris pH 8, 0.5 µL T4 PNK enzyme, 0.5 µL T4 PNK buffer, and 0.5 µL Superasin) at 37°C for 1 hour. Samples were then subjected to SPRI clean up: 50 µL of sample in RNase-free water was added to 90 µL RNAclean beads and 270 µL isopropanol. After washing with 85% ethanol, beads were resuspended in 7 µL RNase-free water. The supernatant was collected, and we proceeded with next-generation sequencing library preparation using the Clontech smRNA library prep kit according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq S2 with single-end 1x50 nt reads. Samples were always processed in large batches of a maximum of 24 samples to avoid sample processing biases. RNA-seq libraries were prepared from 10 ng total RNA using the SMARTer Stranded Total RNA-seq Pico Input Mammalian V2 kit (Takara Bio) according to the manufacturer's instructions. Samples were multiplexed with Illumina TruSeq HT barcodes and sequenced on a NextSeq 2000 with single-end 1x75 nt reads. Samples were always processed in large batches of a maximum of 24 samples to avoid sample processing biases.
GEO
GSM5456557