Trizol

Tissue preparation was done as described by Moura et al. (2018) (link). Briefly, the duodenum mucosa was gently scraped to collect cells. The material recovered consisted mainly of enterocytes, with scattered goblet cells and occasional enteroendocrine cells. In crypts, the material also included Paneth cells and stem cells. After collection, the cells were washed twice in sterile Hank's solution. TRIzol® (Sigma-Aldrich) was then added and the material was then stored at −70 °C.
Mesenteric lymphoid nodules located between the leaflets of the mesentery, from the duodenum to the rectum, were also collected. These were washed twice in sterile Hank’s solution (without Ca and Mg) and macerated. Cells were then collected and suspended in sterile Hank's solution, after which the cell pellets were suspended in TRIzol (Sigma-Aldrich) and stored at −70 °C.
The spleens were removed and macerated, and the splenocytes thus obtained were suspended in a balanced Hank’s solution. Afterward, the cells were centrifuged, and the pellet was suspended in lysis solutions (0.8% ammonium chloride), followed by washing and suspension in sterile Hank's solution (Cultilab, Campinas, Brazil). TRIzol was then added and the material was stored at −70 °C.

600,000 cells were collected, washed twice with 10 mM Tris (pH 7.4) and the cell pellets frozen in liquid nitrogen. Total RNA was extracted with TRIzol (Sigma-Aldrich) following the TRIzol reagent BD protocol.
For RNAseq: library preparation using illumina TruSeq stranded RNA kit and paired-end 2 × 150 bp sequencing on a NovaSeq were performed at the Next Generation Sequencing (NGS) Platform at the University of Bern, Switzerland.
For small RNA seq: small RNAs were size selected by polyacrylamide gel selection (17–35 nt) and library preparation performed using the illumina TruSeq small RNA kit. Single-end 1 × 75 bp sequencing was performed on a NextSeq. Size selection, library preparation and sequencing were performed at Fasteris (Geneva, Switzerland).

RNA was isolated from the IFMs of 2–3-d-old flies. IFMs were removed from the bisected thoraces at 4° and immersed in Trizol (Sigma). Next, RNA was extracted with the help of Trizol (Sigma) as per the manufacturer’s protocol. cDNA was made using 1–2 µg of extracted RNA and a cDNA synthesis kit (Fermentas). The following primers were used: RP49 (FP: 5′-TTCTACCAGCTTCAAGATGAC-3′, RP: -5′-GTGTATTCCGACCACGTTACA-3′); upheld (up) (FP: 5′-CTCGGGTGTCTCGGGCTCAC-3′ RP: 5′-CTCGAACGAGAAGATCTGGA-3′); and Opa1-like (FP: 5′-AACGGTGGAGCCAGTTCTCG-3′; RP: 5′-TGATCTCCGTCTGCAGCGTC-3′). Quantitative PCR was carried out using DyNAmoTM HS SYBR green mix (F-410L; Thermo Scientific). Fluorescence intensities were recorded and analyzed in an ABI Prism 7900HT sequence detection system (SDS 2.1; Applied Biosystems). The relative changes in gene expression were estimated after normalization to the expression of a housekeeping gene, RP49. For semiquantitative PCR, reactions were set up using the 2× PCR Mastermix (Fermentas) and PCR amplification was carried out using a Mastercycler Nexus (Eppendorf).