General methods

2.1.1 Maintenance of human hematopoietic cell lines

Growth conditions. I cultured K562 and Raji B (wildtype or CDK9^as) cells (Table 13) in accordance with the DSMZ Cell Culture standards in RPMI 1640 medium containing 10 % heat inactivated fetal bovine serum (FBS), and L-glutamine or 1 x GlutaMAX supplement at 37 °C in a humidified 5 % CO2 incubator (Table 14). For short term storage, cells were stored at -80 °C. For long term storage, cells were stored in liquid nitrogen. The freezing medium is described in Table 14. For each working stock, a growth curve was generated. Cell morphology was frequently controlled by light microscopy. All three cell lines used in this study displayed the phenotypic properties, including morphology and proliferation rate, that have been described in literature.

Authentication. K562, Raji B wildtype and Raji B (CDK9^as) cell lines were verified by Dr. Wilhelm Dirks (Leibniz Institute DSMZ, Molecular Biology group). DNA profiling was carried out using 18 different and highly polymorphic short tandem repeat (STR) loci. All three cell lines were derived of pure human cell cultures and had full-matching STR profiles, as indicated by a search of databases of cell banks ATCC (USA), JCRB (Japan), RIKEN (Japan), KCLB (Korea), and DSMZ (Germany) (international STR reference database) ⁴⁰⁹. The respective STR electropherograms are shown in Figure 5.

Mycoplasma testing. Tests were performed in-house by Dr. Anna Sawicka (MPI-bpc, Dept.

of Molecular Biology). K562, Raji B wildtype and Raji B (CDK9^as) cells were verified to be free of mycoplasma contamination using Plasmo Test Mycoplasma Detection Kit according to manufacturer’s instructions. The kit provides a cellular assay for colorimetric detection using HEK-Blue™-2 cells in detection medium as sensor.

Cell counting and biological replicates. I counted cells using a hemocytometer (Neubauer counting chamber) in 3 technical replicates, or by a Scepter™ 2.0 Cell Counter (Merck KGaA) according to manufacturer’s instructions. Biological replicates were cultured independently.

Figure 5. STR electropherograms of human hematopoietic cell lines used in this study.

Shown are the STR profiles of human K562 cells (a), Raji B wildtype (b), and Raji B (CDK9^as) (c) cells.

Authentication of cell lines was performed by Dr. Wilhelm Dirks (Leibniz Institute DSMZ, Molecular Biology group). See main text for additional information.

2.1.2 Quantitative reverse transcription PCR of RNA in a two-step assay

I established a qPCR assay to screen for optimal inhibitor concentrations and determine the best time points for subsequent TT-seq, RNA-seq and mNET-seq analysis. This approach makes use of RNA spike-ins for normalization ⁴¹⁰ and targets introns of highly expressed and rapidly degraded transcripts (the selection is based on half-lives measured in K562 cells ¹⁶⁸), or treatment-specific up- or downregulated transcripts (based on literature).

Total RNA isolation and DNase treatment. I harvested 3-4 x 10⁶ cells at 3,000 x g for 2 min for each time point and biological replicate (n>2). Total RNA was isolated with QIAzol according to manufacturer’s instructions except for the addition of 10 ng RNA spike-in mix

168 together with QIAzol. To remove possible genomic DNA contamination, isolated RNA (10 µg) was treated with TURBO DNase according to manufacturer’s instructions.

Reverse transcription (RT). For RT, I used random hexamer priming (5’-d(NNNNNN)-3’, N = G, A, T, or C) according to manufacturer’s instructions. Briefly, 1 µg of DNase-treated RNA, Random Hexamer primers (final concentration of 5 ng/µL), dNTPs mix (final concentration of 0.5 mM) were mixed and incubated at 65 °C for 5 min. Subsequently, Maxima H Minus Reverse Transcriptase (final concentration of 200 U) and 5 x Maxima RT buffer were added (+RT reaction). For DNA contamination control, cDNA synthesis was performed without enzyme (-RT reaction; substituted with water). The (-/+) RT reactions were incubated in a PCR cycler at 25 °C for 10 min, 50 °C for 30 min, and 85 °C for 5 min.

Primer design. I designed primers for quantitative PCR (qPCR) by using the online primer design software Primer3 v.0.4.0 ³⁹⁹. Criteria that differ to default settings were the following:

product size ranges ‘80-150 100-200’; primer size optimum ‘25’; primer Tm minimum ‘60’, optimum ‘64’, maximum ‘70’; maximum Tm difference ‘2’, table of thermodynamic parameters

‘SantaLucia 1998’; product Tm optimum ‘50’; primer GC minimum ‘35’, optimum ‘65’, maximum ‘80’; salt correction formula ‘SantaLucia 1998’; concentration of divalent cations

‘3.5 mM’; concentration of dNTPs ‘0.2 mM’. Primer specificity (single product peak) was validated by melting profiles. Primer sequences, length, annealing temperature, amplicon length and position on target are reported in Table 8.

Quantitative PCR (qPCR). cDNAs (50 ng) were amplified with SYBR® Select Master Mix according to manufacturer’s instruction with a final primer concentration of 400 nM. PCR reactions were run in 96-well optical plates sealed with optical adhesive cover on a qTOWER 2.0/2.2 instrument. The following thermal cycling conditions were used (SYBR Select Master Mix reference, standard cycling mode): 50 °C for 2 min, 95 °C for 2 min, 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Three synthetic RNA spike-ins were used for normalization.

The 2(-ΔΔCt) method was applied to calculate the normalized target gene expression fold change, with the amplification efficiency (E) for each target gene, slope of standard curve (S) and mean threshold cycle (Ct) ⁴¹¹.

2.1.3 Western blotting

I performed Western blotting to determine the quality of cellular fractionation by enrichment of marker proteins in cytoplasm (GAPDH), nucleoplasm (U1 snRNP0), and chromatin (H3/H2B, phosphorylated POLR2A).

The protocol consists of the following steps: sample preparation, protein separation, protein electrotransfer to a membrane, detection of marker proteins including blocking, probing with primary antibody and subsequently, with secondary horseradish peroxidase (HRP) conjugated antibody (subsection 1.4), chemiluminescence detection, and band quantification. Buffer compositions are listed in Table 16.

Protein samples were denatured in 1 x Laemmli buffer at 70 °C for 10 min. Samples were stored at -20 °C, or directly applied to polyacrylamide gel electrophoresis (PAGE).

NuPAGE™ 4-12 % Bis-Tris Protein Gels and MOPS buffer ⁴¹² were used according to manufacturer’s instructions. Briefly, proteins were separated at 200 V for 50 min. A PVDF membrane was cut to the size of the gel, activated in 100 % ethanol for 30 sec, and subsequently equilibrated in transfer buffer. After 50 min, NuPAGE was removed from the cassette, placed onto a filter paper and assembled in a XCell II Blot Module (semi-wet transfer unit) according to manufacturer’s instructions. Transfer of a single NuPAGE onto PVDF membrane was performed in transfer buffer at 30 V for 1 h.

Membrane was stained with Ponceau S staining solution to visualize successful protein transfer ⁴¹³. Membrane blocking was performed in 5 % milk PBS-T on a rocking surface for at least 1 h. Primary antibody was added overnight. A list of primary antibodies used in this study can be found in Table 11. Three washes of the membrane were performed using PBS-T, each with incubation on a rocking surface for 5 min. Secondary antibody was added on a rocking surface for 1 h. A list of HRP-coupled secondary antibodies used in this study can be found in Table 12. Three washes were performed using PBS-T, each with incubation on a rocking surface for 5 min.

ECL working solution of SuperSignal West Pico PLUS Chemiluminescent Substrate was prepared by mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution. After the last PBS-T wash, the membrane was rinsed several times with ECL working solution. Marker proteins were visualized by chemiluminescence detection on INTAS.

Band quantification was performed by Fiji / Image J.

2.1.4 Figures

All figures in this work were generated in Adobe Illustrator. Genome browser views were generated in R/Bioconductor or using the Integrative Genomics Viewer. Metagene plots were generated in R/Bioconductor. Bar plots were generated in Excel.

All plots were subsequently modified for visual purposes in Adobe Illustrator. The respective programs are listed in Table 19.