GSM8565168: TY66_S6; Equus caballus; RNA-Seq
Endometrial biopsies were collected from the base of the uterine horn of pregnant pony mares on days 33 (n=5) and 42 (n=5) post-ovulation, following confirmation of normally progressing pregnancies. Samples were digested to obtain suspensions of single cells according to De Mestre et al. (2010).Cell viability was determined using Trypan blue solution 0.4%. Endometrial cell suspensions were immediately processed for scRNA-seq with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) according to the manufacturer's instructions. An input of 10,000 cells per preparation were used. Libraries were sequenced at a depth of approximately 35,000 paired-end reads per cell on an Illumina NovaSeq6000 (Illumina, San Diego, CA, USA). Library was generated according to the manufacter's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1;Dual Index; 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.