GSM5617608: Induced pluripotent stem cell Iso-T - ATAC_rep2; Homo sapiens; ATAC-seq
RNA was extracted utilizing RNeasy Plus Mini Kit (Qiagen). In situ Hi-C - Hi-C libraries were generated from 2 million iPSCs and NPCs using the Arima Hi-C kit (Arima Genomics, San Diego) following to the manufacturer's instructions without any modifications. Briefly, the Arima Hi-C kit consists of 6 steps: (i) cells were crosslinked with 1% formaldehyde, (ii) DNA was digested using the Arima restriction enzyme cocktail within intact permeabilized nuclei, (iii) ends were filled and biotinylated, (iv) blunt ends were ligated, (v) ligated fragments were purified, sheared and a biotin pull down was performed using streptavidin, and (vi) adapters were ligated, barcoded and amplified using the Kappa Hyperprep Kit (Roche, KK8502).    ChIP-seq - Library preparation consists of 8 steps: (i) cells were crosslinked with 1% formaldehyde, (ii) chromatin was sheared (Covaris E220 Focused-ultrasonicator) after lysing cells, (iii) digested chromatin was incubated with 2ug of LMNB1 antibody (Abcam, ab8982), samples were also incubated without any antibody for input and Spike-in chromatin was incubated with Spike-in antibody (Active motif). (iv) same amount of Spike-in chromatin/antibody was added to each sample. (V) antibody-bound DNA/protein complexes and input samples were captured and washed using Protein G magnetic beads. (vi) cross-links were reversed and protein was digested using proteinase K. (vii) DNA was purified utilizing QIAquick PCR Purification Kit (Qiagen, 28104) (viii) libraries were prepared utilizing the Kappa Hyperprep Kit (Roche, KK8502).    ATAC-seq - Libraries were prepared as previously described (Buenrostro et al., 2015).    RNA-seq - 150 ng of RNA was used to prepare libraries using the Kapa mRNA hyperprep kit for standard RNA libraries (Roche, KK8540) on a TecanEvo 150s (Tecan).