GSM7106558: P2; Homo sapiens; RNA-Seq
Tissue fragments from HCM patients were transfered to the oxygenated Ca2+ free solution and filtered into a conical flask containing enzymatic buffer (275 u/ml collagenase II and 1.2 u/ml protease XXIV). Discard the supernatant once the supernatant become obscure and incubate the fragments in a fresh enzyme solution without protease. Remove the supernatant and re-suspend pelleted myocytes in resuspension buffer. Freshly collected cardiocytes from HCM patients were washed with PBS, picked using piptides under a microscope and then processed for library construction. cDNA libraries were prepared based on the STRT-seq protocol. Briefly, cardiocytes were resuspended in the lysis buffer. 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 barcodes.  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. The pooled barcoded cDNA was then amplified by PCR and fragmented using Tn5 transpose. The apropiated sized fragments were selected with Ampure XP beads