title: GSM3671519: GFP+ sorted cells - bulk RNAseq 3; Homo sapiens; RNA-Seq
library_construction_protocol: Single Cell RNA-seq: around 100 clusters of cells per genotype at day 27 of differentiation are dissociated. Single cells were suspended in PBS+0.04% BSA. Cellular suspensions (~6,000 cells) were loaded on a Chromium Single Cell Instrument (10X Genomics) to generate single cell GEMs. Single-cell RNAseq libraries were prepared using Chromium Single cell 3¢ Library, Gel beads & Mutiplex kit (10X Genomics). Sequencing was performed on Illumina NextSeq500 using the following read length: 59-bp Read1 for transcript read, 14-bp I7 Index for Cell Barcode read, 8-bp I5 Index for sample index read, and 10-bp Read2 for UMI read. The samples were sequenced in ten different libraries (batches). Among all these samples, Batch 980379 and Batch 993091 (2 libraries) are HNF1A mutants (R200Q), Batch 980380 and Batch 993090 (2 libraries) are HNF1A knockouts (KO) while Batch 989516 and Batch 993089 (2 libraries) are HNF1A wildtype cells (WT). Bulk RNA-seq: around 200 clusters of cells are dissociated and sorted for GFP using BD FACS Aria II cell sorter. Sorted cells are pelleted by centrifugation and RNA extracted using total the RNA purification micro (Norgen Biotek, 35300) kit according to the manufactures protocol. RNA quality and concentration is determined by an Agilent bioanalyzer and RNAseq performed by the Columbia University Genome Center. A poly-A pull-down was used to enrich mRNAs from total RNA samples (200ng-1ug per sample, RIN>8 required) and proceed on library preparation by using Illumina TruSeq RNA prep kit. Libraries are then sequenced using an Illumina HiSeq2000.  RTA (Illumina) was used for base calling and bcl2fastq (version 1.8.4) for converting BCL to fastq format, coupled with adaptor trimming.
attributes.tag: GEO Accession
attributes.value: GSM3671519