Bioanalyzer tapestation

It is possible to verify recovery of RNA at this step by spectrophotometry, or more accurately by fluorometry. Integrity of the RNA can be assessed by Bioanalyzer/Tapestation (Agilent) or gel electrophoresis, although the nascent RNA fraction cannot always be visualized by gel electrophoresis due to its comparatively low concentration. In addition, the nascent RNA is refractory to accurate quantification by spectrophotometry because 4sU influences the absorbance spectrum [27 (link)]. The pre-existing fraction is not required for half-life calculations, but can be useful to retain for troubleshooting analysis (see section 3.4). Typical results of Tapestation and fluorometric analyses via the Qubit RNA Broad Range Assay Kit (ThermoFisher Scientific #Q10210) for two sets of fractionated samples are shown in Figure 4. Note that the concentration of the nascent RNA fraction is generally less than that for the pre-existing and total RNA fractions but it remains intact and gives an acceptable RIN. The abundance of rRNA in the eluate is generally lower than in the total RNA fraction due to the fact that rRNA has a relatively long half life.

ChIP-Seq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1 ng of DNA according to the manufacturer’s protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by a Qubit fluorometer and samples were QC’D using a Bioanalyzer Tapestation (Agilent Technologies 2200) to determine fragment size. Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on a NovaSeq targeting 40 million 100bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute.

Tumors were minced thoroughly and digested by 0.5 mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies). scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per μl (Single Cell 3’ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flow cell. Average cell recovery for Prkci^f/fPrkcz^f/f;Villin-Cre tumors was 104,954 cells with a total of 209,907 cells captured at a mean depth of 14,568 read per cell and 895 mean genes per cell.

Tumors were minced thoroughly and digested by 0.5 mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies). scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per μl (Single Cell 3′ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). On average, approximately 8,000 cells were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flow cell. Average cell recovery for the orthotopic in the Fsp1-cre mice was 23,576 cells, with a total of 47153 cells captured at a mean depth of 18712 reads per cell and 1224 mean genes per cell. Average cell recovery for the orthotopic in caecum was 15628 cells, with a total of 46886 cells captured at a mean depth of 18712 reads per cell and 1492 mean genes per cell.