title: GSM7817780: snRNA-seq of LAND-treated glioblastoma nuclei with 10x Multiome (DEFND-seq RNA library) - PTO037; Homo sapiens; RNA-Seq
library_construction_protocol: snATAC-seq: Nuclei were prepared from cryopreserved BJ fibroblasts. Frozen cells were thawed and added to a Dounce homogenizer containing 1 mL of nuclei isolation buffer (NIB: 10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal (Millipore Sigma #18896), and 1 x protease inhibitors (Millipore Sigma #118735800001)). The sample was gently homogenized on ice with 5 strokes of the loose pestle and 5 strokes of the tight pestle. The cells were transferred to a conical and 5 mL of PBS (Gibco #10010-031) was added. The sample was spun down at 500 g for 5 minutes, washed with 10 mL of PBS and resuspended in 100 µL of 10x Genomics Nuclei Buffer (10x Genomics, #2000207).  xSDS snDNA-seq: Nuclei that were cross-linked and then treated with sodium dodecyl sulfate (SDS) to remove nucleosomes (xSDS) were prepared following Adey et al. Briefly, BJ fibroblasts were washed with 10 mL of PBS and covered in 3 mL of TrypLE (ThermoFisher #12605028) for 10 minutes at 37 °C. Then 9 mL of media was added, quenching the dissociation, and cells were spun down. The cells were resuspended in 10 mL of media with 406 µL of 37% formaldehyde (Millipore Sigma #252549) and gently rotated at room temperature for 10 minutes. 2.5 M glycine (800 µL) was then added and the suspension was incubated on ice for 5 minutes. Cells were centrifuged at 500 g for 8 minutes and washed with 10 mL of PBS. 5 mL of NIB was added to the pellet and the suspension was incubated for 20 minutes at 4 °C under rotation. Cells were then spun down at 500 g for 5 minutes and washed with 900 µL of NEBuffer 2.1 (New England Biolabs #B7202). The cells were pelleted (500 g for 5 minutes) and resuspended in NEBuffer 2.1 with 12 µL of 20% SDS. The solution was placed in a gentleMACS and vigorously shaken at 42 °C for 30 minutes. 200 µL of 10 % Triton-X (Millipore Sigma #T8787) was added and the cells were again shaken at 42 °C for 30 minutes.  LAND snDNA-seq/DEFND-seq: The LAND protocol for adherent cells using 10x reagents was adapted from the LAND protocol. BJ fibroblasts were first washed with 10 mL of PBS and dissociated with 3 mL of TrypLE for 10 minutes at 37 C. Media (9 mL) was used to quench the dissociation reaction and collect the cells. The cells were spun down (300 g for 5 minutes) and 200 µL of DEFND buffer (175 µL NIB, 10 µL 1 mg/mL protease inhibitor (Millipore Sigma #11429868001), 25 µL 100 mM lithium diiodosalicylate (Millipore Sigma #653-14-5)), was added and incubated on ice for 5 minutes. Immediately following this incubation, 10 mL of nuclei isolation buffer was added and the nuclei were centrifuged at 4 °C for 5 minutes at 500 g. The supernatant was removed and 100 µL of 10x Genomics Nuclei Buffer. A fraction of nuclei was stained with SYBR green and counted on a Countess with a GFP filter set.  GBM Cryopreserved Tissue DEFND-seq: A cryopreserved IDH wildtype Grade IV GBM sample was obtained from excess material collected for clinical purposes from de-identified brain tumor specimens. Nuclei were prepared from this anonymous sample according to XXXXX . Frozen tissue was placed in a Dounce homogenizer containing 1 mL of homogenization buffer (HB: 1 µM DTT, 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM tris, 1x protease inhibitor, 0.4 U/µL RNAse, 0.2 U/µL SUPERase In) and subjected to 15 strokes using the loose piston and 15 strokes using the tight piston. The homogenate was divided into 5 volumes and each was strained twice. The filtered homogenate was combined into a tube and centrifuged at 1000 g for 10 minutes at 4 °C. The pellet was resuspended in 1000 µL of HB and strained with 4 strainers (250 µL of homogenate for each strainer). The strained homogenate was again combined into a tube and centrifuged at 1000 g for 10 minutes at 4 °C. The pellet was resuspended in 250 µL of HB to which 250 µL of 50% iodixanol dilution media (IDM: 250 mM sucrose, 150 mM KCl, 30 mM MgCl2, 60 mM tris) was added resulting in a 25% IDM/nuclei suspension. This suspension was carefully layered on top of a tube containing 50% IDM at the bottom and 29% IDM at the surface, and centrifuged at 18,000 g for 23 minutes at 4 C. The nuclei formed a white pellet or sheet in the middle of the tube between the layers. All solution around this pellet was removed – first from the top and then from the bottom, and the pellet was resuspended in 1 mL of chilled PBS and centrifuged (500 g for 5 minutes at 4 °C). The supernatant was removed and the pellet was then treated with DEFND buffer for 5 minutes on ice. Immediately following this incubation, 5 mL of NIB with RNAse inhibitors was added at the nuclei were centrifuged at 500 g for 5 minutes at 4 °C. snATAC-seq: Single-cell ATAC libraries were prepared with these nuclei using the 10x Genomics Single Cell ATAC kit (10x Genomics #1000176, #1000162, and #1000212) according to the manufacturer's protocol. xSDS snDNA-seq: The cells were pelleted (500 g for 5 minutes) and resuspended in 100 µL of 10x Genomics Nuclei Buffer, counted, and tagmented following the Chromium Single Cell ATAC protocol. Tagmented nuclei were loaded onto a 10x Chromoium controller and libraries were prepared according to the Chromimum Single Cell ATAC protocol (10x Genomics #1000176, #1000162, and #1000212). LAND snDNA-seq: Nuclei were then tagmented and prepared for single-cell sequencing with the Chromium Single Cell ATAC kit (10x Genomics #1000176, #1000162, and #1000212) and protocol. DEFND-seq: Nuclei were then tagmented and prepared for single-cell sequencing with the Chromium Single Cell Multiome kit (10x Genomics #1000285, #1000230, #1000215). DEFND-seq/10x Multiome (snRNA/DNA-seq), snATAC-seq, snDNA-seq