GSM5661075: F116-ATAC; Mus musculus; ATAC-seq
A single cell suspension of ESCs and EpiLCs were harvested at a concentration of 1 million cells/mL; 1 million cells were lysed and RNA extracted using RNeasy (Qiagen) RNA extraction kit; 100,000 cells were used for transposition reaction. For ChIP, 10 million cells were harvested and nuclei isolated using hypotonic lysis. Chromatin was fragmented using Covaris followed by dialysis and immunoprecipitation against protein targets was performed. For single cell RNAseq, single cell suspension was achieved for EB cultures and 500,000 cells for each individual sample was labeled with unique Multiseq LMO barcode. Labelled samples were pooled at 3,000 cells per sample and 40,000 cells were loaded onto a single lane of a 10X Chromium microfluidic chip. Cells were lysed in droplets using 10X Chromium instrument and reverse transcription was performed in droplet and library construction performed according to 10X Chromium v3 chemistry. Libraries were constructed using the KAPA mRNA HyperPrep Kit for ESC and EpiLC bulk RNA samples. Modified Illumina Nextera adapters were used to construct ATAC libraries. ChIP-seq librariers were constructed using KAPA HyperPrep Kit. Single cell RNA-seq library preparation was performed using 10X Chromium v3.