title: Single cell short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
design: Single cell short- and long-read RNA-sequencing of iNeurons in Fragile-X and control cell-lines
library_name: IsoB11_sc_ONT_rep3_s
library_construction_protocol: NGN2-induced glutamatergic neurons were generated from Fragile X syndrome patient-derived induced pluripotent stem cells (iPSCs) and an isogenic CRISPR-corrected rescue line. Neuronal differentiation was performed for 21 days using doxycycline-inducible NGN2 expression. Cells were cultured on polyornithine/laminin coated plates and maintained with BDNF-containing neuronal medium. Cells were harvested at day 21 for downstream single-cell and bulk RNA sequencing. For single-cell RNA-seq, cell viability was confirmed to exceed 95% prior to processing. Approximately 6,000 cells per replicate were targeted using the 10x Genomics Chromium Next GEM Single Cell 3' Kit v3.1. Following Post-GEM-RT Cleanup and cDNA amplification, the resulting cDNA was utilized for all downstream short- and long-read library preparations. Single-cell libraries were generated using the 10x Genomics Chromium platform with the NextGEM Single Cell 3′ v3.1 kit following the manufacturer's protocol. Approximately 6,000 cells were loaded per channel to generate barcoded single-cell cDNA. Following reverse transcription and cDNA amplification, libraries were prepared for Illumina sequencing via enzymatic fragmentation, end repair, A-tailing, and adapter ligation. Unique i5 and i7 indices were incorporated using the Dual Index Kit TT Set A. Libraries were validated using an Agilent TapeStation D1000. 10 ng of single-cell cDNA generated from the 10x Genomics Chromium NextGEM Single-Cell 3′ Kit v3.1 workflow was used as input. Amplified cDNA was processed according to the Oxford Nanopore single-cell transcriptomics protocol (SST_v9198_v114_revE), including pull-down of biotin-tagged cDNA using M280 magnetic streptavidin-coated beads and a second PCR round using PRM primers. Following QC, libraries were prepared using the Ligation Sequencing Kit V14 (SQK-LSK114). Amplified single-cell cDNA (75 ng) from the 10x Genomics Chromium NextGEM Single-Cell 3′ Kit v3.1 workflow was used for PacBio library preparation across two sequencing rounds. Libraries were generated using the MAS-Seq for 10x 3' Concatenation Kit (PB, 102-407-900) or the Kinnex Single-Cell RNA Kit (PB, 103-072-200) according to the manufacturer's instructions. SMRTbell libraries were purified with SMRTbell Cleanup Beads (PB, 102-158-300).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-16805:IsoB11_sc_ONT_rep3_s