Instrument: Illumina Genome Analyzer
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Frozen, isolated CA1 hippocampal slices were thawed in ice-cold homogenization buffer (20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100μg/ml CHX (cycloheximide), 25U/ml Turbo DNaseI (Ambion, #AM2238), 1X EDTA-free protease inhibitor (Roche), avoiding detergent in nuclease-free water) on ice for 5min. Wide orifice tips were used to transfer slices to a pre-chilled detergent-free Dounce homogenizer. Tissues were slowly homogenized by hand (20 strokes of loose pestle A, and 20 strokes of tight pestle B). Homogenates were carefully transferred to clean 1.5ml tubes with clean glass Pasteur pipets and bulbs. 1% NP-40 was added to the homogenates and incubated on ice for 10min. Homogenates were clarified by centrifugation at 2,000g 4 °C for 10min. The supernatants were collected and clarified again by centrifugation at 20,000g 4 °C for 10min. The supernatants were collected, and the amounts of nucleic acid were measured by Nanodrop (A260 units). For each sample, cytoplasmic RNA for RNA-seq was purified from one-fourth of the lysate with TRIzol LS reagent (Invitrogen, #10296028). The other three-fourths of the lysate was digested with 100ng RNase A (Sigma, # R4875) and 60U RNase T1 (Thermo Fisher Scientific, #EN0542) per A260 at 25°C for 30 min and stopped by chilling on ice and adding 50U SUPERase In RNase inhibitor (Ambion, #AM2694). Digested lysates were applied to 10%-50% (w/v) sucrose gradients prepared in 1X polysome buffer (20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100μg/ml CHX in nuclease-free water). After the ultracentrifugation in a SW41Ti rotor (Beckman Coulter) at 35,000 rpm (avg 151,263g) 4°C for 2.5 hours, gradients were fractionated at 1.5 ml/min and 12 sec collection intervals through a fractionation system (Brandel) that continually monitored A260 values. Monosome fractions were identified, pooled, and extracted with TRIzol LS. 3' ligation based library construction, which is similar to miRNA-seq. Ribosome protected mRNA fragments were ligated to 3' adapters. RT was then performed and cDNA was circularized. PCR was performed to add adapters for Illumina sequencing. In parallel, input RNA samples were processed similarly as the ribosome footprints except the following steps. After rRNA depletion, input RNA mixed with an equal volume of 2x alkaline fragmentation solution (2 mM EDTA, 10 mM Na2CO3, 90 mM NaHCO3, pH ~ 9.3) and heated at 95°C for 15min to achieve an average fragment length of ~140nt. Fragmented RNA samples were separated on a 10% TBU gel and size-selected between the 100 and 150nt markers. RT products were separated on a 10% TBU gel and the 200-250nt region was selected. Antisense probe depletion was omitted for input RNA samples. 260-300bp final PCR products were size-selected on an 8% TBE gel. The size distributions of final libraries were measured by Fragment Analyzer (Advanced Analytical, performed by Molecular Biology Core Labs at UMMS). The concentrations were quantified with KAPA Library Quantification Kit (Kapa Biosystems, #KK4835). Libraries were pooled with equal molar ratios, denatured, diluted, and sequenced with NextSeq 500/550 High Output Kit v2 (Illumina, 75bp single-end runs, #FC-404-2005) on a Nextseq500 sequencer (Illumina).