title: GSM6433674: bacTRAP HFD INPUT 3; Mus musculus; RNA-Seq
library_construction_protocol: BacTrap-based ribosomal profiling of hypothalamic ECs  Afﬁnity puriﬁcation of translating ribosomes (Trap) was performed as described previously (Heiman et al., 2014; 2008)  with minor modiﬁcations. 12-week-old male and female Slco1c1CreERT2tg/wt::ROSA26lSlL10aGFPtg/wt mice were used for the BacTrap experiment (an equal number of male and female mouse hypothalamic tissue was pooled in each sample). 3 weeks after tamoxifen induction, mice were put either on a CD or an HFD for 3 days. Afterwards, mice were sacrificed and the hypothalamus was rapidly dissected using a stainless steel brain matrix (Agnthos) and immediately frozen in liquid nitrogen. Buffer preparation: Lysis buffer (20mM HEPES [pH 7.4], 5mM MgCl2, 150 mM KCl, 0.5 mM DTT, 100µg/ml cycloheximide, 40U/ml Ribonuclease Inhibitor (Rnasin, Promega) and protease inhibitor [1 tablet of cOmplete mini EDTA-free protease inhibitor cocktail/7 mL and 2 tablets of PhosSTOP/10 ml]) ; 0.15M KCl IP wash buffer (20mM HEPES [pH 7.4], 5mM MgCl2, 150 mM KCl, 1% NP-40, 0.5 mM DTT, 100 µg/ml cycloheximide) ; 0.35 M KCl IP wash buffer (20mM HEPES [pH 7.4], 5mM MgCl2, 350 mM KCl, 1% NP-40, 0.5mM DTT, 100 µg/ml cycloheximide). Bead preparation: Dynabeads Protein A (375 µl, Novex, per IP) were washed in 1mL 0.15M KCl IP wash buffer for three times. Afterward, beads were resuspended in 275µL 0.15M KCl IP wash buffer. 50 µg of antibody 19C8 and 50 µg of antibody 19F7 (Cat# Htz-GFP-19F7, RRID: AB_2716736 and Htz-GFP-19C8, RRID: AB_2716737, Memorial Sloan Kettering Monoclonal Antibody Facility) per IP was added into each bead tube. The beads were incubated with slow end-over-end mixing at 4°C overnight. Afterward, the supernatant was removed and the beads were washed in 1mL 0.15 KCl IP wash buffer for 3 three times. After the wash, beads were resuspended in 200µL 0.15M KCl IP wash buffer. Pooled hypothalamic tissue (4 pooled mice per sample; 4 samples per group) was homogenized on ice with lysis buffer (1mL per sample) at 4 °C. Homogenates were transferred to low binding microcentrifuge tubes (Nonstick, RNase-free microfuge tubes,1.5 ml, Ambion, Invitrogen) and centrifuged 10 minutes, 2000g at 4 °C to obtain the post-nuclear supernatant. The supernatant was transferred to a new tube on ice, and 1/9 sample volume of 10% NP-40 (ﬁnal concentration: 1%) and 1/9 sample volume of 300 mM DHPC (ﬁnal concentration: 30 mM) were added. This solution was mixed, incubated on ice for 2 min and then centrifuged at 17,000 g for 10 min at 4 °C. The resulting supernatant was transferred to a new tube and a 25 µL aliquot was removed, transferred to a new tube, ﬂash frozen in liquid nitrogen and stored at -80°C for puriﬁcation as input RNA. 200 µL of antibody-bound beads were added (800-1000 µL supernatant) and incubated at 4°C for one hour with end-over-end mixing. Beads were collected with a magnet and resuspended in 1 mL 0.35 M KCl IP wash buffer. Beads were washed three more times with 1mL of 0.35 M KCl IP wash buffer. After the ﬁnal wash, the beads were collected with a magnet and the supernatant removed. The RNA was eluted by the addition of buffer RLT (350 µL) to the beads, allowed to incubate at room temperature (RT) for 5 minutes and RNA was subsequently puriﬁed using the RNeasy Micro Kit (#74004, QIAGEN). RNA integrity was assessed using an Agilent 2100 bioanalyzer.     Single nucleus isolation and sorting   Single nucleus sequencing was performed according to Habib et al. (2017) with minor modiﬁcations. Hypothalami were dissected and homogenized in 2ml EZ PREP (#NUC101, Merck) on ice and incubated for 5 min with an additional 2 ml ice-cold EZ PREP. The nuclei were centrifuged at 500g for 5 min at 4 °C. The pellets were washed with 4 ml ice-cold EZ PREP and incubated on ice for 5 min. After centrifugation, the nuclei were washed in 4ml nucleus suspension buffer (NSB, consisting of 1× PBS, 0.04% BSA and 0.1% RNase inhibitor (#2313A, Clontech)). Isolated nuclei were resuspended in nucleus suspension buffer (400µl per hypothalamus) and filtered through a 35μm prewetted cell strainer. The nuclei suspension was stained with DAPI (1:1000, #62248, Thermo Fisher Scientiﬁc). ZsGreen/DAPI double-positive nuclei were collected as brain endothelial cell nuclei. BD FACSAria IIIu was used for gating and sorting nuclei. Single nuclei were identified based on side scatter (SSC-A) and DAPI fluorescence (area versus width). ZsGreen fluorescence was detected with 488 nm excitation and 530 nm emission. Sorting was performed at 4 °C with a 70 µm nozzle. Sorted ZsGreen positive nuclei were collected into 1xPBS buffer with 0.04% BSA for 10x genomics single nucleus sequencing. RNA Bulk Sequencing   Pre-ampliﬁcation using the Ovation RNASeq System V2 was performed. Total RNA was used for ﬁrst strand cDNA synthesis, using both poly(T) and random primers, followed by second strand synthesis and isothermal strand-displacement ampliﬁcation. For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used, with 1 ng cDNA input. After validation (Agilent 2200 TapeStation) and quantiﬁcation (Invitrogen Qubit System) all 24 transcriptome libraries were pooled. The pool was quantiﬁed using the Peqlab KAPA Library Quantiﬁcation Kit and the Applied Biosystems 7900HT Sequence Detection and sequenced on an Illumina NovaSeq S2 ﬂowcell with a PE100 protocol. The RNA sequencing pipeline utilizes the GRCm38 assembly of the mouse genome as gene sets from Ensembl release 96 (Yates et al., 2015). We quantiﬁed the gene expression of each sample by (1) aligning the RNA-sequencing reads to the mm10 reference genome using hisat 2.1.0 (Kim et al., 2015). (2) computationally depleted rRNA reads using samtools 1.8.0 (Li et al., 2009) and (3) transcript assembly and quantiﬁcation as well as the differential analysis between the experimental conditions using the cufﬂinks 2.2.1 suite (Trapnell et al., 2010).