Hemocytes from unfed Amblyomma americanum
Single cell samples were assessed for proper quality controlmetrics prior to library preparation. All samples were quantitatedthrough the Invitrogen Countess 3 FL Automated Cell Counter(Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) usingReadyCount Green/Red Viability Stain (ReadyCount Green/RedViability Stain) per manufactures protocol with GFP filter. Sampleswere all greater than 90% viable with cell density averaging 1000cells/ul. A total of 10000 cells per sample were used for downstreamlibrary preparation. Single cell samples were processed through the10X Genomics Chromium Single Cell 3 protocol v3.1 permanufactures instruction. Single cell droplets (GEMs) wereproduced using the 10X Genomics Chromium Controller andbarcoded for sample identification. Final libraries were validatedon the Qiagen QIAxcel DNA High Sensitivity Bioanalyzer(QIAGEN, Germantown, MD, USA)), to visualize proper insertsize, and concentration was calculated using the KAPA IlluminaQuantification Kit (Kapa Biosystems, Wilmington, MA, USA) onthe Bio-Rad CFX96 thermal cycler (Bio-Rad Inc., Hercules, CA,USA). Pooled libraries were sequenced on the Illumina NextSeq2000 per 10X Genomics specifications (28x91 cycles).