GSM2501158: LCM_fasting_basal_3; Mus musculus; RNA-Seq
Tissue blocks for microdissection were obtained from 8-week-old male B6 mice.The small intestine was briefly washed in cold PBS and embedded and frozen in OCT without fixation. 8µm thick sections were cut from the frozen block, mounted on polyethylene-naphthalate membrane-coated glass slides (Zeiss, A4151909081000), air-dried for 1m at room temperature, washed in 70% ethanol (30s), incubated in water (Sigma, W4502, 30s), stained with HistoGene Staining Solution (ThermoFisher Scientific, KIT0401, 20s), washed vigorously in water for a total of 30s. The stained sections were dehydrated with subsequent 30s incubations in 70%,95% and 100% ethanol and air dried for 3m before microdissection.  Tissue sections were microdissected on a UV laser-based PALM Microbeam (Zeiss). The system makes use of a pulsed UV laser that cuts the tissue at indicated marks with minimal damage to surrounding cells; the cutting was performed with the following parameters: PALM 40X lens, cut energy 48 (1-100), cut focus 69 (1-100).  Tissue fragments were catapulted and collected in 0.2ml adhesive cap tubes (Zeiss, A4151909191000) with these settings: LPC energy 57 (1-100), LPC focus 67 (1-100). The capturing success was visually confirmed by focusing the PALM on the targeted adhesive cap after each use of the laser-catapult. A total of 15’000 µm2 of subcellular apical or basal tissue areas was collected for RNA sequencing experiments. Library preparation for microdissected tissues was achieved by resuspension of the fragments in 9.5µl H2O and 1µl of 10x reaction buffer of the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, 634888) in the adhesive cap of the collection tubes. Tissue lysis was achieved by incubation for 5m at room temperature; the lysed samples were flash frozen until library preparation. The RNA was amplified with the SMART-Seq v4 kit according to the manufacturer’s instructions and by using 15 PCR cycles for the cDNA amplification step. The amplified cDNA was converted into sequencing library with the Nextera XT DNA Library kit (Illumina, FC-131-1024). 