GSM6935728: Gemcitabine + I-BRD9 scRNA-seq; Homo sapiens; RNA-Seq
Chromatin complexes were eluted from the beads by incubation with ChIP elution buffer (1% SDS+TE) at 65 °C for 1 hr. After that, proteinase K (Life Technologies, cat.#AM2548) was added to the elution reverse cross-linked chromatin complexes for overnight. After centrifugation at 16,000g for 1 hour at 4°C, DNA pellets were washed once with ice-cold 70% ethanol, and finally air dried. ChIP samples were resuspended in 30µl and 1:10 of the samples were used  in  qPCR for verifying the ChIP samples. Cells were centrifuged at 300g for 3 min, the supernatant aspirated, the cell pellet resuspended in 150 ul of Isotonic Lysis Buffer (10 mM Tris-HCl pH 7.5, 3 mM CaCl, 2 mM MgCl2, 0.32 M Sucrose and Protease Inhibitors, Roche), and incubated for 12 min on ice. Triton X-100 from a 10% stock was then added at a final concentration of 0.5%, the samples were vortexed briefly and incubated on ice for 6 min. The samples were centrifuged for 5 min at 400g at 4°C, and the cytoplasmic fraction removed from the nuclear pellet. The samples were resuspended gently in 625uL of PBS and transferred to a fresh 1.5 ml eppendorf tube. The nuclei were centrifuged at 1500g for 3 min at 4°C and the supernatant aspirated thoroughly from the nuclear pellet. This step was immediately followed by tagmentation (Nextera DNA Sample Preparation Kit for 24 Samples, FC-121-1030) by resuspending each sample in 100 µL Nextera mastermix (52.5 µl TD buffer, 42.5 µl of water and 5 µl of TDE1 per reaction). The nuclear pellet was resuspended thoroughly by pipetting and incubated at 37 °C for 1 hour shaking at 300rpm. Total RNA was isolated by RNeasy RNA Extraction Kit (Qiagen) according to manufacturer's guidelines. RNA was then eluted in 30µl of water and the concentration was measured using Nanodrop. The master mix was prepared as follows: 8µl 5x First-Strand Buffer (Invitrogen), 0.5µl Random primers (0.5 ug/ml) (Promega Cat. C1181), 1ul dNTP mix (10 mM each) (Promega Cat.U1515), 2 ul 0.1 M DTT, 0.5 ul RNase Out, 0.25 ul Superscript III Reverse Transcriptase (Life Technologies). 500 ng of total RNA into a separate tube with 11.75 ul   RNase-free water. RNA was heated to 65 ⁰C for 5 min and allowed to chill on ice for 2 min. 8.25 ul of the master mix were added to RNA. The reaction was incubated at 25 ⁰C for 10 min and then at 42⁰C for 50 min. The reaction was then inactivated by heating at 70 for 15 min. After collection of cells, centrifuge at a speed not exceeding 400rcf (adjust the speed and centrifugation time according to the cell types). Carefully discard the supernatant and retain the cell precipitation. The cell pellet was resuspended by 1mL 1X PBS containing 0.04% BSA and the washing procedure was repeated twice. After washing, appropriate volume PBS was added to the cell precipitation to obtain single-cell dispersion suspension with a concentration close to the goal number. A type of wide-bore pipette tip was used in cell resuspension for a lower cell damage. If necessary, cell filter should be applied to remove cell debris and clusters (eg. 30μm MACS SmartStrainers and 40μm FlowmiTM Cell Strainer). Automatic cytometry was used to determine the cell concentration. And the sample volume was calculated based on the optimal cell sampling concentration supplied by 10X official website and the target capture number. If the calculated concentration is too high, adjust the liquid volume to the appropriate concentration and repeat counting once more. Single-cell mRNA libraries were built using the Chromium Next GEM Single Cell 3' Library Construction V3 Kit (10X Genomics) according to manufacturer's protocol.