GSM9864779: HCI-005, DMSO, MN93, ATAC; Homo sapiens; ATAC-seq
After 3 weeks, organoids were dissociated with TrypLE for 10 minutes until reached a single-cell suspension. Single cell suspension was counted and between 400.000 and 1.000.000 cells were frozen in PDxO medium (70%) plus FBS (20%) and DMSO (10%). Single cell nuclei were isolated using the Chromium Nuclei Isolation Kit with RNase Inhibitor (10x Genomics, PN-1000494), Chromium Nuclei Isolation Reagents Kit (10x Genomics, PN-1000447) and Chromium Nuclei Isolation Consumables Kit (10x Genomics, PN-1000448). Cryopreserved PDxO were transferred to 2 mL tubes, centrifuged (4°C /2 min/1200g) and washed one time with 1.7 ml PBS then spun again (4°C /2min/1200g). All supernatant was removed, preserving the pellet and samples were kept samples on ice. Lysis buffer was prepared by 1:10 diluting the lysis buffer from 10x Genomics Nuclei isolation kit (freshly prepared as in 10x Genomics manual CG000505, Rev B, 1X+10%: Lysis Reagent, 550 µl, Reducing Agent B, 0.55 µl and Surfactant A 5.5 µl). The diluent was buffer containing 10mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2. Samples were lysed using 200 µl lysis buffer for 4-10 min. depending on the input cell number (400K-2M cells). 1 ml Wash Buffer (1% BSA in PBS with RNase inhibitor (10x Genomics)) was added to stop the lysis, and samples were centrifuge at 4°C /7 min/600g, supernatant removed preserving the pellet. The nuclei pellet was resuspended in 1X Nuclei Buffer (10x Genomics). The volume varied from 50-200 µl/sample depending on the size of the nuclei pellet to achieve a target concentration of 3500 nuclei/µl. For RREMS, genomic DNA was extracted from leftover nuclei pellets sent for multiome using the DNeasy Blood & Tissue Kit (Qiagen, Cat no. 69504). Single-nucleus chromatin accessibility and gene expression libraries were prepared using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression platform (10x Genomics, Pleasanton, CA, USA) following the manufacturer's protocol (Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide, CG000338 Rev. G). For RREMS, 100ng of genomic DNA is digested with MspI (NEB) to enrich for CG rich regions of the genome. DNA fragment overhangs are filled with Klenow Fragment (3'-5' exo-) (NEB) to leave an A-overhang. Next, we ligated the NEBNext® EM-seq™ Adaptor (NEB, E7165) and then samples were purified using AmpureXP beads (1:2X). To convert unmethylated cytosine to uracil we followed the NEB protocol for the NEBNext® Enzymatic Methyl-seq v2 Conversion Module (E8020L) using formamide at the step 1.7 (Denaturation of DNA). The converted libraries were then PCR amplified using Epimark HS Taq DNA Polymerase (NEB, M0490L) and the barcoded primers NEBNext® Multiplex Oligos for Enzymatic Methyl-seq (Unique Dual Index Primer Pairs Plate, E7166). PCR cycling conditions were 95°C for 30 sec, (94°C for 30 sec, 60°C for 3 mins, for 14 cycles). Libraries were purified using AmpureXP beads (1:1.2X). Equimolar quantities of barcoded libraries were pooled and sequenced using the Illumina NovaSeq X instrument. PhiX genome control library (Illumina) was included at 20% in each sequencing lane to improve cluster identification and base-balancing during sequencing.