Wta2 kit

Ten thousand HSCs or HPCs were double sorted directly into RLT plus RNA lysis buffer (Qiagen) and RNA was isolated with RNAeasy micro plus columns (Qiagen). Transcripts were amplified with the WTA2 kit (Sigma) with the Kreatech ULS RNA labeling kit (Kreatech Diagnostics). Labeled cDNA was hybridized to Agilent Mouse 8 × 60 K microarrays and analyzed with an Agilent C-class scanner. Signal data were assembled and processed in Partek RRID:SCR_011860, and samples were compared by Linear Models for Microarrays, RRID:SCR_010943 (Ritchie et al., 2015 (link); Smyth, 2004 (link)). Adjusted p-values were calculated by the Benjamini and Hochberg false discovery rate (Benjamini and Hochberg, 1995 ). Z-scores were calculated as previously described (Cheadle et al., 2003 (link)). Hierarchical cluster analysis was performed with Cluster 3.0 and visualized with Java TreeView; RRID:SCR_013505 and RRID:SCR_013503 (Eisen et al., 1998 (link)). Principal component analyses and Euclidean distance comparisons (by permutation testing) were performed with the R software environment. Microarray data sets have been deposited into Gene Expression Omnibus (GSE81153). GSEA was performed using gene sets that were generated as cited in the text, or with gene sets curated in the MSigDB databases; RRID:SCR_003199 (Subramanian et al., 2005 (link)).

Total RNA was amplified by the WTA2 kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's protocol. cDNAs were chemically labeled with Kreatech ULS RNA Labeling Kit (Kreatech Diagnostics, Durham, NC, USA). Labeled cDNA was purified with Qiagen PCR purification columns, and cDNAs were quantitated on a NanoDrop spectrophotometer. cDNA samples were hybridized to Agilent Mouse_v1 8x60K microarrays (Design ID-028005, Agilent Technologies, Santa Clara, CA, USA). Slides were scanned on an Agilent C-class Microarray scanner to detect Cy5 fluorescence, according to the manufacturer's specifications. Gridding and analysis of images was performed using Feature Extraction software (v11.5.1.1, Agilent Technologies, Santa Clara, CA, USA). To identify differentially expressed genes in each hypothalamic nucleus, ratios of signal intensities to each hypothalamic nucleus were calculated, and genes whose expression levels were greater than or equal to 10-fold in each hypothalamic nucleus compared to the others were selected. The heat map for each log2-transformed gene expression level was generated using Partek Genomics Suite (Partek Inc., St. Louis, MO, USA). The microarray data were deposited into the NCBI GEO database (GEO Accession Number GSE58238).

Approximately 10⁷ MEFs were resuspended in hypotonic buffer (20 mM Hepes-KOH pH 7.9, 10 mMKCl, 1 mM MgCl₂, 0.5% NP40, 20% Glycerol) and dounced 25 times on ice. The run-on transcription assay was performed as described previously (38 (link)). Run-on RNA was reverse transcribed using whole transcriptome amplification (WTA2) kit according to the manufacturer (Sigma Aldrich), and the resultant cDNA was labeled with Cy3 or Cy5 and hybridized to the same custom tiling arrays used for hybridizing ChIP DNA.

All 254 fecal samples were prepared for high-throughput virome sequencing using the NetoVIR protocol (75 (link)). In short, each fecal aliquot was homogenized in phosphate-buffered saline (PBS) (30 mass/volume percentage), centrifuged, filtered (0.8 μm), and subjected to nuclease treatment to enrich for viral-like particles. Next, the QIAamp viral RNA minikit (Qiagen) without carrier RNA was used to extract both RNA and DNA. The extracts were reverse transcribed and randomly amplified (17 cycles) using a modified WTA2 kit (Sigma-Aldrich). Sequencing libraries were prepared with the Nextera XT DNA library preparation kit (Illumina) and sequenced on the NextSeq 500 high-throughput Illumina platform (Nucleomics Core facility, KU Leuven, Belgium). Per sample, a median of 12.1 million (IQR, 6.9 million to 19.4 million) paired-end reads (2 × 150 bp) were generated.

RNA was extracted from thawed cell pellets with the Direct-zol RNA miniprep kit (Zymogen) including DNase treatment according to the manufacturer’s instructions. The WTA2 kit (Sigma) was used to generate a cDNA library according to the protocol provided by the manufacturer (25 cycles), and PCR was performed with the PerfeCTa SYBR green FastMix (Quanta Biosciences) using a Roche LightCycler 480.

Library preparation and amplification were performed according to the manufacturer's instructions for the WTA2 kit (Sigma-Aldrich). In the first step of the WTA2 protocol, the RNA was reverse transcribed implementing non self-complementary and quasi-random 3′ and universal 5′ primers. The newly synthetized single strands were the templates for annealing and extension steps. When the SYBR Green signal reached a plateau, the reaction was stopped. The resultant library contained cDNA fragments with size distribution between 100 and 1000 base pairs (data obtained from Agilent Bioanalyzer using DNA 1000 microfluidic chip; Supplementary Figure 2B). The amplified double-stranded cDNA was purified and quantified on a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).