GSM9100442: 10x Multiome with D0, D2, D4 and D7 of iPSC to HPC differentiation in one lane. GEX; Homo sapiens; RNA-Seq
hiPSC (WTC11) with the dCas9-KRAB was differentiated into hepatic progenitor cells over 7 days of differentiation. We seeded 550,000 cells per well in 6-well plates in StemFlex media, Normocin with Rock Inhibitor and 1   g/ml Doxycycline. Next day, we changed the media with fresh StemFlex, Normocin and Doxycycline. The following day, we collected the cells from some wells and froze them in CryoStor media (STEMCELL Technologies); these were denoted as Day 0 (D0) cells. For the remaining wells, we changed the media with fresh RPMI (Thermo), B27 supplement (Thermo), 4 uM CHIR99021 (Reprocell), Normocin and Doxycycline. Next day, we changed the media with fresh RPMI, B27, Normocin and Doxycycline. The following day, we collected the cells from some wells and froze them in CryoStor media; these were denoted as Day 2 (D2) cells. For the remaining wells, we changed the media with fresh Hepatic Differentiation Media consisting of Knockout DMEM (Thermo), 20% knockout serum replacement (Thermo), 2 mM GlutaMax (Thermo), 100 uM 2-Mercaptoethanol (Sigma), 1x MEM Non-essential amino acids (Thermo), 1% DMSO (Sigma), along with Normocin and Doxycycline. The cells were incubated for 2 days after which we collected the cells from some of the wells and froze them in CryoStor media; these were denoted as Day 4 (D4) cells. For the remaining wells, we changed the media with fresh Hepatic Differentiation Media with Normocin and Doxycycline. The cells were incubated for 2 days after which the media was again changed with fresh Hepatic Differentiation Media with Normocin and Doxycycline. The following day, we collected the cells from all the wells and froze them in CryoStor media; these were denoted as Day 7 (D7) cells. For the multiome experiment, we thawed one vial each of D0, D2, D4 and D7 cells and centrifuged at 300 x g for 5 min, then resuspended in 1 ml of chilled PBS with 0.04% BSA. We washed the cells again in PBS with 0.04% BSA and proceeded with nuclei isolation from 0.5-1 million cells. We centrifuged the cells at 500 x g for 5 min at 4  C, removed the supernatant and resuspended in 100 ul of chilled 1x lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin, 1% BSA, 1 mM DTT, 1U/ul Sigma Protector RNase Inhibitor). We incubated the cells for 4 mins on ice and then added 1 ml of chilled wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 0.2U/ul Thermo RNaseOUT, 0.2U/ul Takara Recombinant RNase Inhibitor). We centrifuged the nuclei at 500 x g for 5 min at 4  C, removed the supernatant and resuspended in 1 ml of chilled wash buffer. After counting we combined nuclei for the D0-D7 samples, centrifuged at 500 x g for 5 min at 4  C, resuspended in diluted nuclei buffer (1x Nuclei buffer, 1U/ul Sigma Protector RNase Inhibitor, 1 mM DTT), and adjusted the concentration to ~7300 nuclei/ul. We proceeded with transposition and library preparation using the 10x Genomics Multiome kit (PN-1000284), loading the sample onto one well of the multiome chip for GEM generation. ATAC and gene expression (GEX) libraries were prepared according to the 10x Genomics protocol.