C1 single cell auto prep ifc chips

For N. brasiliensis, three small (5–10 μm) C1 Single-Cell Auto Prep IFC chips (Fluidigm) were primed and 5000 cells were sorted directly into the chip. For P. chabaudi, cells were loaded at a concentration of 1700 cells μl^-1 onto C1 small chips. To allow estimation of technical variability, 1 μl of a 1:4000 dilution of ERCC (External RNA Controls Consortium) spike-in mix (Ambion, Life Technologies) was added to the lysis reagent. Cell capture sites were visually inspected one by one using a microscope. The capture efficiency is described in Additional file 2: Tables S1 and S7.
The capture sites that did not contain single cells were noted and were removed from downstream analysis. Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech) and the Advantage 2 PCR kit according to the manufacturer’s instructions on the C1 device. cDNA was harvested and diluted to 0.1–0.3 ng/μl and libraries were prepared in 96-well plates using a Nextera XT DNA Sample Preparation kit (Illumina) according to the protocol supplied by Fluidigm. Libraries were pooled and sequenced on an Illumina HiSeq2500 using paired-end 75-bp reads for N. brasiliensis and 100-bp reads for P. chabaudi.

For S. mansoni, three small (5–10 μm) C1 Single-Cell Auto Prep IFC chips (Fluidigm) were primed and 5000 cells were sorted directly into the chip. To allow estimation of technical variability, 1 μl of a 1:4000 dilution of ERCC (External RNA Controls Consortium) spike-in mix (Ambion, Life Technologies) was added to the lysis reagent. Cell capture sites were visually inspected one by one using a microscope. The capture sites that did not contain single cells were noted and were removed from downstream analysis. Reverse transcription and cDNA preamplification were performed using the SMARTer Ultra Low RNA kit (Clontech) and the Advantage 2 PCR kit according to the manufacturer’s instructions on the C1 device. cDNA was harvested and diluted to 0.1–0.3 ng/μl and libraries were prepared in 96-well plates using a Nextera XT DNA Sample Preparation kit (Illumina) according to the protocol supplied by Fluidigm. Libraries were pooled and sequenced on an Illumina HiSeq2500 using paired-end 75-bp reads.
Salmon was used to estimate gene expression counts [23 (link)]. Poor quality libraries were eliminated using Scater [24 (link)] based on exonic and mitochondrial read counts. For all queries, a gene was considered expressed if the Log10 (1+normalized count) was above 0.5. Gene overlap was tested using Fisher’s method. Counts and condition matrices are provided in S2 File.