biostudies-arrayexpressHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14102Proof of principle and testing experiments on  iPS2-sci-seq have been performed on two batches of hPSC-derived cardiomyocytes at day 23. Cells have been treated with Tetracycline throughout differentiation. For this experiment it was used a 2-level of indexing sci-RNAseq protocol (Cao J. et. al, Science 2017). During RT and PCR indexing steps, additional primers have been used to enrich for UCI and shRNA barcodes for the iPS2-seq pipeline. Sequencing has been performed on a NextSeq 500 with a High Output kit 75 cycles. Read1, 18 cycles; Index1, 10 cycles; Index2, 10 cycles, Read2, 52 cycles.biostudies-arrayexpressSample Collection - hPSC tranfected in pool with iPS-2seq plasmids in AAVS1 locus (multiple perturbations in pool). shRNA expression has been induced throughout differentiation with Tetracycline. hPSC derived cardiomyocytes were cultured for 23 days with tetracycline. Then Harvested at single cell suspension and processed for nuclear extraction.Nucleic Acid Extraction - Nuclear extraction by IGEPAL 0.3% ipotonic buffer. PFA fixation. RNAse inhibitors have been used during all steps to avoid RNA degradation.Sequencing - Next seq 500 75 cycles high output. Read1, 18 cycles; Index1, 10 cycles; Index2, 10 cycles, Read2, 52 cyclesLibrary Construction - 2-level indexing sci-RNAseq protocol for the gene expression library and the barcode library. shRNA barcodes have been enriched during RT and PCR indexing by a nested PCR binding the TET repressor.MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesData Transformation - RT barcode sequences are atached to allow demultiplexing of the second level indexing. First level of indexing have been demultiplexed with the Illumina Bcl2fastq platform and the samplesheet atached (which includes the PCR indexing and the well of indexing). This generates fastq files per each well containing around 25 cells inside.Data Transformation - Processed data and demultiplexed to single cell with CatcheR (association to the RT indexing barcode is still needed) and associated with a shRNA perturbation with CatcheR_barcode function. Atached is the txt files with the cell annotation and counts, then also a RDS file that can be read with the readRDS() function in R ans subsequently processed with monocle 3.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of coding RNA from single cellsHomo sapiensERP160365Elisa BalmasfalseiPS2-sci-sec dataset on hPSC derived cardiomyocytes at day 23Proof of principle and testing experiments on  iPS2-sci-seq have been performed on two batches of hPSC-derived cardiomyocytes at day 23. Cells have been treated with Tetracycline throughout differentiation. For this experiment it was used a 2-level of indexing sci-RNAseq protocol (Cao J. et. al, Science 2017). During RT and PCR indexing steps, additional primers have been used to enrich for UCI and shRNA barcodes for the iPS2-seq pipeline. Sequencing has been performed on a NextSeq 500 with a High Output kit 75 cycles. Read1, 18 cycles; Index1, 10 cycles; Index2, 10 cycles, Read2, 52 cycles.2025-10-27T00:00:00Z2025-10-27T09:34:27.685Z2024-05-17T16:51:16.049ZE-MTAB-14102ERP160365EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184