title: GSM4471639: HCT-116_SP1-HyPBase; Homo sapiens; OTHER
library_construction_protocol: Total RNA was isolated from each replicate using the RNEasy Plus Mini Kit following manufacturer's instructions. Briefly, cell pellets were resuspended in 600 µl of Buffer RLT Plus with 1% 2-mercaptoethanol. Cells were homogenized by vortexing. DNA was removed by running lysate through gDNA Eliminator spin columns, while RNA was bound by passing the flow-through over RNEasy spin columns. An on-column treatment with DNaseI was also performed. After washing, RNA was eluted in 40 µl RNase-free H2O. RNA was quantitated using the Qubit RNA HS Assay Kit. We performed first strand synthesis on each replicate with Maxima H Minus Reverse Transcriptase. We mixed 2 µg of total RNA with 1 µl 10 mM dNTPs and 1 µl of 50 µM SMART_dT18VN primer (for a complete list of oligonucleotides, see Table S5), brought the total volume up to 14 µl, and incubated it at 65ºC for 5 minutes. After transferring to ice and letting rest for 1 minute, we added 4 µl 5X Maxima RT Buffer, 1 µl RNaseOUT, and 1 µl of 1:1 Maxima H Minus Reverse Transcriptase diluted in 1x RT Buffer (100 U). The solution was mixed by pipetting and incubated at 50ºC for 1 hour followed by heat inactivation at 85ºC for 10 minutes. Finally, we digested with 1 µl RNaseH at 37ºC for 30 minutes. cDNA was stored at -20ºC. The PCR conditions for amplifying self-reporting transcripts (i.e. transcripts derived from self-reporting transposons) involved mixing 1 µl cDNA template with 12.5 µl Kapa HiFi HotStart ReadyMix, 0.5 µl 25 µM SMART primer, and either 1 µl of 25 µM SRT_PAC_F1 primer (in the case of puromycin selection) or 0.5 µl of 25 µM SRT_tdTomato_F1 primer (in the case of tdTomato screening). The mixture was brought up to 25 µl with ddH2O. Thermocycling parameters were as follows: 95ºC for 3 minutes; 20 cycles of: 98ºC for 20 seconds–65ºC for 30 seconds–72ºC for 5 minutes; 72ºC for 10 minutes; hold at 4ºC forever. As a control, cDNA quality can be assessed with exon-spanning primers for β-actin (see Table S5 for examples of human primers (Raff et al., 1997)) under the same thermocycling settings. PCR products were purified using AMPure XP beads. 12 µl of resuspended beads were added to the 25 µl PCR product and mixed homogenously by pipetting. After a 5-minute incubation at room temperature, the solution was placed on a magnetic rack for 2 minutes. The supernatant was aspirated and discarded. The pellet was washed twice with 200 µl of 70% ethanol (incubated for 30 seconds each time), discarding the supernatant each time. The pellet was left to dry at room temperature for 2 minutes. To elute, we added 20 µl ddH2O to the pellet, resuspended by pipetting, incubated at room temperature for 2 minutes, and placed on a magnetic rack for one minute. Once clear, the solution was transferred to a clean 1.5 ml tube. DNA concentration was measured on the Qubit 3.0 Fluorometer using the dsDNA High Sensitivity Assay Kit. Calling card libraries from bulk RNA were generated using the Nextera XT DNA Library Preparation Kit. One nanogram of PCR product was resuspended in 5 µl ddH2O. To this mixture we added 10 µl Tagment DNA (TD) Buffer and 5 µl Amplicon Tagment Mix (ATM). After pipetting to mix, we incubated the solution in a thermocycler preheated to 55ºC. The tagmentation reaction was halted by adding 5 µl Neutralization Tagment (NT) Buffer and was kept at room temperature for 5 minutes. The final PCR was set up by adding 15 µl Nextera PCR Mix (NPM), 8 µl ddH2O, 1 µl of 10 µM transposon primer (e.g. OM-PB-NNN) and 1 µl Nextera N7 indexed primer. The transposon primer anneals to the end of the transposon terminal repeat–piggyBac, in the case of OM-PB primers, or Sleeping Beauty, in the case of OM-SB primers–and contains a 3 base pair barcode sequence. Every N7 primer contains a unique index sequence that is demultiplexed by the sequencer. Each replicate was assigned a unique combination of barcoded transposon primer and indexed N7 primer, enabling precise identification of each library's sequencing reads. The final PCR was run under the following conditions: 95ºC for 30 seconds; 13 cycles of: 95ºC for 10 seconds–50ºC for 30 seconds–72ºC for 30 seconds; 72ºC for 5 minutes; hold at 4ºC forever. After PCR, the final library was purified using 30 µl (0.6x) AMPure XP beads, as described above. The library was eluted in 11 µl ddH2O and quantitated on an Agilent TapeStation 4200 System using the High Sensitivity D1000 ScreenTape.
attributes.tag: GEO Accession
attributes.value: GSM4471639