Illumina NovaSeq 6000 paired end sequencing; Single cell RNA sequencing of iPSC and Human dermal fibroblasts
Single cell RNA sequencing of iPSC and Human dermal fibroblasts
Freshly prepared iPSC and DMFB cells were loaded onto the Chromium™ Controller (10x Genomics®) on different days. Cell number and viability were measured by Countess™ II Automated Cell Counter (Thermo Fisher®). Final cell density was adjusted to 1.0×106cells/ml with >95% viability. Both cells were targeting ~5,000 cells per reaction. For sc-end3-dT libraries, we used Chromium™ Single Cell 3′ Library kit v2 (10x Genomics®). Briefly, single cell suspensions were mixed with the Single cell Master Mix using Reverse transcription (RT) Primer (AAGCAGTGGTATCAACGCAGAGTACATr-GrGrG) and loaded together with 3′ gel beads and partitioning oil into a Single Cell A Chips according to the manufacturer's instructions (10x Genomics®). For sc-end5-dT and sc-end5-rand libraries, used Single Cell 5′ Library kit v1.1 (10x Genomics®). Single cell suspension was mixed with Single cell Master Mix using oligo(dT) RT primer (AAGCAGTGGTATCAACGCAGAGTACGAGAC-T(30)-VN) or random hexamer RT primer (AAGCAGTGGTATCAACGCAGAGTACNNNNNN) and loaded together with 5′ gel beads and partitioning oil into a Single Cell A Chips according to the manufacturer's instructions. RNAs within single cells were uniquely barcoded and reverse transcribed within droplets. Both methods used Veriti™ Thermal Cycler (Applied Biosystems®) for RT reaction.  After collecting cDNAs prepared from each method, they were amplified using cDNA primer mix from the kit, followed by the standard steps according to manufacturer's instructions. For iPSC and DMFB, six libraries (i.e. 3 methods × 2 cell lines) were barcoded by different indexes from i7 sample index plate (10x Genomics®). The libraries were examined in Bioanalyzer™ (Agilent®) for size profiles and quantified by KAPA™ Library Quantification Kits (Kapa Biosystems®)