Ribozero depletion kit

We depleted the purified RNA of ribosomal RNA using the Illumina RiboZero depletion kit. Depleted RNA was submitted to the MIT BioMicro Center for strand‐specific library preparation. We ran the libraries on a HiSeq2000 with paired‐end reads and a read length of 40 nucleotides. BWA was applied to map the reads to the V. cholerae type strain, using unique mapping reads. Any read that overlapped with any region of a gene was counted as a read. We then employed DEseq analysis to identify significant changes in gene expression between cells grown in the presence and absence of mucin glycans.

We extracted total RNA from 20 pairs of ovaries of females aged 3–5 days per replicate per genotype. For each genotype (HOAP[mel] and HOAP[yak]), we prepared three biological replicates for a total of six samples. We dissected ovaries into cold 1XPBS and proceeded with RNA extraction using the standard Trizol-based protocol (Invitrogen, Carlsbad, CA). To remove DNA contamination, we used TURBO DNase (Thermo Fisher Scientific, Waltham, MA) then purified samples using a Qiagen RNeasy kit (Qiagen, Hilden, DE). For total mRNA sequencing, the Weill Cornell Epigenetics Core performed ribosomal RNA depletion using Ribo-Zero depletion kit (Illumina, San Diego, CA) and prepared libraries using the Illumina TruSeq Stranded Total RNA kit (Illumina, San Diego, CA). The Core sequenced 200 ng per sample on a HiSeq 2500 using SBS kit v4 on a single-end flow cell (50 cycles). For small RNA sequencing, Fasteris SA (Geneva, CH) conducted acrylamide gel size selection and anti-2S treatment with proprietary oligos as well as standard library preparation protocols using an Illumina TruSeq small RNA kit. They sequenced the libraries on an Illumina NextSeq500 (run mode 1 × 50).