GSM2942566
GSM2942566: mouse_a_v2; Mus musculus; RNA-Seq
Tissue blocks for microdissection were obtained from 8-12 week-old male C57BL/6 mice. The proximal Jejunum 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-Aldrich, 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 20X lens, cut energy 48 (1-100), cut focus 65 (1-100). Tissue fragments were catapulted and collected in 0.2ml adhesive cap tubes (Zeiss, A4151909191000) with these settings: LPC energy 67 (1-100), LPC focus 67 (1-100). The capturing success was visually confirmed by focusing the PALM on the targeted adhesive cap after the collection session. 8-10 Villi above 500µm length selected for microdissection for each of three mice, their epithelium was divided into 5 segments of equal length and isolated.  A total of 30'000-45'000 μm2 of villus epithelium area was collected for each of the five villus zones per mouse. Library preparation for microdissected tissues was performed based on a previously published protocol (https://doi.org/10.1126/science.aan2399) with minor modifications. Specifically, we resuspended microdissected 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. 1ng of the amplified cDNA was converted into sequencing library with the Nextera XT DNA Library kit (Illumina, FC-131-1024). The quality control of the resulting libraries was performed with an Agilent High Sensitivity D1000 ScreenTape System (Agilent, 5067- 5584). Libraries that passed quality control were loaded with a concentration of 1.8pM on 75 cycle high output flow cells (Illumina, FC-404-2005) and sequenced on a NextSeq 500 (Illumina) with the following cycle distribution: 8bp index 1, 8bp index 2, 38bp read 1, 38bp read 2.
GEO
GSM2942566
GEO Accession
GSM2942566