NextSeq 2000 sequencing; PROseq (Precision Run-On sequencing) to capture nascent RNA of wild type and mutant mouse embryonic stem cells
PROseq (Precision Run-On sequencing) to capture nascent RNA of wild type and mutant mouse embryonic stem cells
Mouse ES cells were cultured on 0.2% gelatin-coated plates in ES medium (DMEM, supplemented with 15% FBS, LIF, 2-Mercaptoethanol, 2 mM L-Glutamine and 1x non-essential amino acids) at 37°C and 5% CO2. Medium was changed daily and cells were split every second day. ES cell permeabilization was performed on ice for 10 min and followed by 1 wash with the permeabilization buffer and centrifugations at 1000x g at 4°C. Cells were resuspended in storage buffer in appropriate aliquots, snap-frozen and stored at -80°C until further usage. For the experiment, 10MIO cells were used per sample with a spike-in of 0.5MIO permeabilized Drosophila KC cells. The nuclear run-on reaction was performed as a 2 biotin run-on. For this, the 2x Nuclear-run on master mix was mixed with biotin-11-UTP, biotin-11-CTP, ATP, GTP, H2O, RNase inhibitor and Sarkosyl. The reaction mix was pre-heated at 37°C and the appropriate number of cells added to each reaction vial, mixed thoroughly and incubated at 37°C for 5 min. To stop the reaction, 700 μl of the RNA Lysis Buffer from the ZYMO Quick-RNA Miniprep kit were added and vortexed. RNA extraction was performed according to the kits manual. Final RNA was eluted 2x with 50 μl H2O and pooled to a final volume of 100 μl. Precision Run-On sequencing (PRO-seq) was performed as described in (Mahat et al., 2016, doi:10.1038/nprot.2016.086) with some minor changes. Mouse embryonic stem (ES) cells were harvested using ice-cold Trypsin-EDTA, washed 2x with cold PBS and centrifugations at 200x g at 4°C. Permeabilization was performed on ice for 10 min and followed by 1 wash with the permeabilization buffer and centrifugations at 1000x g at 4°C. Cells were resuspended in storage buffer in appropriate aliquots, snap-frozen and stored at -80°C until further usage. For the experiment, 10MIO cells were used per sample with a spike-in of 0.5MIO permeabilized Drosophila KC cells (5%). The nuclear run-on reaction was performed as a 2 biotin run-on. For this, the 2x Nuclear-run on master mix was mixed with biotin-11-UTP, biotin-11-CTP, ATP, GTP, H2O, RNase inhibitor and Sarkosyl. The reaction mix was pre-heated at 37°C and the appropriate number of cells added to each reaction vial, mixed thoroughly and incubated at 37°C for 5 min. To stop the reaction, 700 μl of the RNA Lysis Buffer from the ZYMO Quick-RNA Miniprep kit were added and vortexed. RNA extraction was performed according to the kits manual. Final RNA was eluted 2x with 50 μl H2O and pooled to a final volume of 100 μl. For the base hydrolysis, the RNA was denatured for 40s at 65°C and snap-cooled on ice. Ice-cold 1N NaOH was added and incubated on ice for 10 min. RNA was precipitated, washed, air-dried and resuspended in 50 μl H2O plus 1 μl RNase Inhibitor. Biotin RNA enrichment was performed by adding 50 μl of pre-washed streptavidin beads and incubation on a rotator set to 8 rpm at RT for 20 min. Beads were washed 2x with ice-cold high-salt buffer, 2x ice-cold binding buffer and 1x low-salt buffer before the RNA was cleaned up using Trizol and cholorform. For the 3' RNA adaptor ligation, the RNA pellet was dissolved in 4 μl of VRA3 3' RNA adaptor dilution, heat-denatured at 65°C for 20s and placed on ice. 6 μl of the RNA ligation mix using T4 RNA ligase was added and incubated at 20°C for 4 hours and kept at 4°C overnight. The next day, a second biotin RNA enrichment was performed and the final RNA pellet dissolved in 5 μl H2O. RNA was heat-denatured at 65°C for 20s and snap-cooled on ice. For the 5' cap repair reaction, the RNA was mixed with the enzyme mix containing RppH and ThermoPol Reaction Buffer and incubated at 37°C for 1 hour. For the following hydroxyl repair, 90 μl of the PNK mix were added and incubated at 37°C for 1 hour. The RNA was cleaned-up using Trizol and chloroform. For the 5' RNA adaptor ligation, the RNA pellet was dissolved in 4 μl of VRA3 3'RNA adaptor dilution, heat-denatured at 65°C for 20s and placed on ice. 6 μl of the RNA ligation mix using T4 RNA ligase was added and incubated at 20°C for 4 hours and kept at 4°C overnight. The next day, a third biotin RNA enrichment was performed and the final RNA pellet dissolved in 10 μl H2O. The RNA was reverse transcribed using the Superscript IV transcriptase and the RP1 reverse-transcription primer. The correct number of PCR cycles was determined by test PCR and Bioanalyzer analysis. In the end, the final PCR was performed using 14 cycles and the final library was cleaned-up using magnetic SPRI beads at a ratio of 1.8x and further size selected with a SPRI beads ratio of 1.3x. The final library was run on a NextSeq 2000 P3 with 50 bp single-end.