GSM5452297: HEK_10_TT_Seq repeat2; Homo sapiens; RNA-Seq
Cells were washed rapidly in PBS before adding 0.7 mL QIAZOL per 10 million cells. The cells were scraped off into 2mL Eppendorffs, vortexed for 10 s and placed on ice for 10 minutes. If required for comparative calibration, 2.4 ng of thio-labelled RNA spike-ins per 1 million cells were added at this stage. Total RNA was purified by adding 320 µL chloroform, vortexing for 10 s, incubating at room temperature for 3 minutes, and centrifugation at 4 ◦C for 15 minutes at 12000 rpm. The upper phase was transferred to a new Eppendorf with 800 uL isopropanol. The sample was left at room temperature for 5 minutes before centrifugation at 4 ◦C for 15 minutes at 15000 rpm. After removing the supernatant, the pellet was washed 3 times in 320 uL 75% ethanol (prepared freshly), with centrifugation at 4 ◦C for 15 minutes at 15000 rpm. The pellet was then air-dried for 10 minutes before dissolving in 160 µL RNAse-free water. At this stage samples were frozen at -80 ◦C. RNA amounts were checked with Nanodrop. 300 µg of total RNA in 400 µL water were chilled on ice for 10 minutes. Samples were sonicated in a Bioruptor at the 'HIGH' setting for 1 cycle at 30s on / 30 s off. Samples were split equally into two Eppendorfs, incubated at 65 ◦C for 10 minutes, and then on ice for 5 minutes. After quick-spinning, 300 µL RNase-free water, 100 µL 10x Biotinylation Buffer (100 mM Tris pH 7.5, 1m mM EDTA pH 8.0), 200 µL DMF, and 200 mL EZ-link HPDP Biotin in DMF (1mg / mL, stored in the dark at 4 ◦C). Incubate in the dark at room temperature for 2 hours with shaking at 800 rpm. RNA was purified using Phase-Lock tubes and resuspended in 100 µL RNase-free water. Samples were pooled back together. For thio-labelled RNA separation, a wash buffer (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 1 M NaCl, 0.1 % Tween-20) was prepared freshly – one kept at room temperature (RT-WB), and one at 65 ◦C (65-WB). 100 µL uMACS streptavidin beads were added to 200 µL RNA-Biotin and incubated in a thermomixer at 4 ◦C for 15 minutes at 800 rpm. During this time, the uMACS magnetic stand and rack were prepared alongside a drainer. uMACS columns were placed into the magnetic rack and equilibrated with 900 µL RT-WB for 10 minutes. The biotin-RNA-bead mix was added to the uMACS column. The flow-through was collected and re-loaded to the columns. The second flow-through was discarded. Columns were washed 3 times with 900 µL of 65-WB and then 3 times with 900 µL RT- WB. Elution of labelled RNA was carried out with 100 µL 100 mM DTT. After 5 minutes, elution was performed again with an additional 100 uL 100 mM DTT. Samples were then immediately carried through for RNA purification using the miRNeasy kit. RNA was eluted with 15 µL RNase-free water. Quality and quantity of thiolabelled RNA was checked using the Agilent Bioanalyzer with an RNA pico chip. Sequencing libraries were prepared using the NEBNext rRNA Depletion kit for Human Cells followed by the NEBNext II Ultra Directional RNA Library prep kit, following both kit instructions. Library quality was assessed using the Agilent Bioanalyzer with a DNA-High Sensitivity chip. Pooled libraries were sequenced on the Illumina NextSeq 500 platform using the High-Output Illumina Nextseq 500 kit. Thiolabelled spike-ins were generated by amplifying 3 1000-bp long DNA frag- ments from genomic yeast DNA with a 42 % GC content, covering the genes bat2, hxt1, and gal1 using forward primers containing the T7 promoter sequence. The PCR product was purified and then transcribed in vitro following the MEGAscript T7 tran- scription kit instructions, using a thio-UTP to UTP ratio of 1:5.