title: GSM3229463: CA2CB496; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted using the QIAGEN micro RNeasy RNA extraction kit (cat #74004). Approximated 20 LCM caps (5 sections/cap) were acquired per region (8 regions per brain) and immediately lysed with 10ul of lysis buffer incubated at room temperature for 5min. Lysates were stored at -80C until further processing. For RNA extraction, lysates from each subregion were pooled and RNA was isolated one at a time to minimize degradation. All samples were subjected to on-column DNase-treatment (Qiagen, cat#79254). We obtained 2-30ng of high quality total RNA (RIN >8.7) from each subregion and compartment, measured using the Agilent RNA 6000 Pico kit and 2100 Bioanalyzer. cDNA libraries were made using the Ovation Universal RNA-Seq system for mouse (NuGEN, part # 0348) according to the manufacturer's instructions, including the shearing step to 200bp with a Covaris S-series Sonication System, using 1ng total RNA input for dendrite samples and 5ng total RNA input for cell body samples. Samples from each mouse (8 samples/mouse, cell body and dendrite samples from each subregion, CA1, CA2, CA3 and DG) were processed together without the experimenter blind to sample IDs. The resulting cDNA libraries were amplified using 16 cycles of PCR prior to quantification using the High Sensitivity DNA kit (Agilent, Cat # 5067-4626) and a 2100 Agilent Bioanalyzer, as well as fluorometrically using Qubit dsDNA HS Assay kit and a Qubit 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). Libraries from one animal were eight-plexed and sequenced on one high throughput lane on an Illumina NextSeq 500 instrument acquiring 100 bp paired-ends reads to a depth of 50 million reads per sample (+/- 10 mil). Four samples (out of 24 samples total) had fewer than 60% of paired-end reads mapped, therefore their libraries were remade and four-plexed on an additional mid-throughput lane as described above.
attributes.tag: GEO Accession
attributes.value: GSM3229463