title: GSM8000358: suprachiasmatic nucleus, control, 20, replicate 3, female, read 1; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted form liver samples using standard Trizol procedure (Invitrogen). Brains were cryosectionned (Leica CM3050, head at -15°C chamber -20°C), first by coarsely trimming the anterior brain until the start of the SCN, then by cutting 23 sequential 25-micrometer coronal sections, each immediately mounted onto a single PEN Membrane Glass Slide (LCM0522, ThermoFisher) and allowed to dry for a few seconds. Slides with mounted sections were warmed at room temperature for a few seconds, then fixed for 3 min in ethanol:acetic acid (19:1), rinsed briefly by immersion in water, stained by immersion in water with 0.2% toluidine blue followed by two washes by immersion in water. All solutions were ice-cold and RNase-free. After the last wash, excess water was flicked off from the slide and slide was placed (sections face up!) onto a plate heater at 50°C for a few minutes until dry. On the same day, SCN were microdissected using a laser dissection microscope (Leica LMD7000; 10× magnification), using 350 microliter RLT buffer with 1% b-mercaptoethanol as a collection/lysis buffer (RNeasy micro kit, Qiagen). RNA was extracted following manufacturer's instructions (RNeasy micro kit, Qiagen). Total RNA was submitted to the Genomic Technologies Core Facility (GTCF). Quality and integrity of the RNA samples were assessed using a 4200 TapeStation (Agilent Technologies) and then libraries generated using the Illumina® Stranded mRNA Prep. Ligation kit (Illumina, Inc.) according to the manufacturer's protocol. Briefly, total RNA (typically 0.025-1ug) was used as input material from which polyadenylated mRNA was purified using poly-T, oligo-attached, magnetic beads. Next, the mRNA was fragmented under elevated temperature and then reverse transcribed into first strand cDNA using random hexamer primers and in the presence of Actinomycin D (thus improving strand specificity whilst mitigating spurious DNA-dependent synthesis). Following removal of the template RNA, second strand cDNA was then synthesized to yield blunt-ended, double-stranded cDNA fragments. Strand specificity was maintained by the incorporation of deoxyuridine triphosphate (dUTP) in place of dTTP to quench the second strand during subsequent amplification. Following a single adenine (A) base addition, adapters with a corresponding, complementary thymine (T) overhang were ligated to the cDNA fragments. Pre-index anchors were then ligated to the ends of the double-stranded cDNA fragments to prepare them for dual indexing. A subsequent PCR amplification step was then used to add the index adapter sequences to create the final cDNA library. The adapter indices enabled the multiplexing of the libraries, which were pooled prior to cluster generation using a cBot instrument. The loaded flow-cell was then paired-end sequenced (76 + 76 cycles, plus indices) on an Illumina HiSeq4000 instrument. Finally, the output data was demultiplexed and BCL-to-Fastq conversion performed using Illumina's bcl2fastq software, version 2.20.0.422