Transcriptomic profiling of LAMA5-deficient human stem cell–derived chondrogenesis reveals a human-specific ECM–WNT signaling axis
Transcriptomic profiling of LAMA5-deficient human stem cell–derived chondrogenesis reveals a human-specific ECM–WNT signaling axis
Cells were harvested directly from in-vitro culture plates. Culture medium was aspirated, and cells were washed with PBS. Cells were lysed immediately using lysis buffer for RNA extraction. Not applicable; samples are cell lines derived from in-vitro culture. Urine-derived stem cells (USCs) were expanded in proliferation medium at 37°C in a humidified 5% CO2 atmosphere. For differentiation experiments, cells were cultured in specific Osteogenic or Chondrogenic differentiation media. Samples designated for differentiation were treated with either Osteogenic or Chondrogenic induction media. For the subset of KO2 samples testing Lithium Chloride, cells were treated with LiCl or left untreated (vehicle control) prior to harvest. Raw data was converted into reads and demultiplexed using Illumina Dragen Software (v. 3.8.4). Unwanted RNA was filtered from the reads using bwa mem (version 0.7.17-r1188) in combination with samtools (version 1.17) and converted back into fastq format using the SamToFastq tool from GATK (version 4.2.1.0). Total RNA was extracted from cells using [Qiagen RNeasy Mini Kit] following the manufacturer's instructions. RNA quality and concentration were assessed using a Fragment Analyzer [Agilent]. Sequencing libraries were prepared using two distinct protocols depending on the sample batch.  For Poly-A enrichment samples: Libraries were prepared using the Illumina Stranded mRNA Prep kit, utilizing Oligo(dT) beads for mRNA capture. For Depletion samples: Libraries were prepared using the Twist RNA Library Prep with Depletion kit, utilizing enzymatic depletion for rRNA and Globin removal. Both protocols generated strand-specific (first-strand) paired-end libraries.