Gene expression is estimated with the EM estimation algorithm and reported as TPM (Transcripts Per Million) and RPKM (Reads per kilobase of transcript per million mapped reads) using CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights). Raw FASTQ formatted sequence reads were imported into CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights)Īdaptor sequences and bases with low quality were trimmed and reads were mapped to the reference genome (GRCh38.102) using the RNAseq analysis tool with the default parameters recommended for RNAseq analysis in CLC Genomics Workbench (version 20.0.2, QIAGEN Digital Insights). RNA libraries were prepared and sequenced by BGI using DNB-seq PE100 platform. RNA samples were collected in 2mL RNAse-free tubes and chilled on ice throughout the procedure. Total RNA was isolated using Direct-zol RNA MicroPrep Kit (Zymo Research) according to the manufacturer’s protocol. The dissolved Matrigel was removed by rinsing 3 times in cold PBS. Matrigel was dissolved by incubating the hCOs in chilled Cell Recovery Solution (Corning, cat. 2013, Nature).įor total RNA extraction the following samples were collected from three independent culture plates: 10 pooled human cerebral organids (hCO) for day 10 samples, 6 pooled hCOs for day 20 samples, and 4 pooled hCOs for day 40 samples. After 5 days all hCOs were transferred into low-attachment 24-wells plates (1 hCO/well) and the remainder of the previously described whole brain organoid protocol was followed (Lancaster et al. All cells and hCOs were maintained in a humid incubator at 37 ☌ with 5 % CO2. Media was changed every 12 hours for the first 3 days with fresh hESC medium with basic fibroblast growth factor (bFGF) (Invitrogen) and ROCK inhibitor (Y-27632, LC laboratories) for all hCOs (microwell and 96-well). After 5 minutes, 2.5 mL media was slowly added to the wells avoiding the disruption of the seeded cells in the microwells. The device was gently placed into the incubator for 5 minutes to allow cells to settle into the microwells. For each microwell device, 60 µL of hESC suspension at 32,000 cells/well was pipetted into the microwell enclosure using a cut p200 tip. Number of cells seeded per well was experimentally optimized for successful generation of embryos bodies (EB) in microwells. 96-well and microwell human cerebral organoids (hCOs) were generated using the same pool of hESCs and differentiated using the same protocol as previously described (Lancaster et al. Cells were maintained in tissue culture dishes (Fisher Scientific Corning Costar) coated with 0.5 mg/cm2 Vitronectin (VTN-N) (Thermo Fisher Scientific) in E8 medium (Thermo Fisher Scientific) and passaged using standard protocols. GEO help: Mouse over screen elements for information.Ĭell line: H9 human embryonic stem cells (WA09, WiCell)Įmbryoid body formation method: Agarose microwell shape roundįeeder-independent H9 human embryonic stem cells (hESCs) (WA09) were obtained from WiCell.
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