GSM9719014: U2OS WT 24h replicate BR2; Homo sapiens; OTHER
Total RNA from the transfected cells preserved in RNAlater was extracted using the RNeasy Mini Kit (Qiagen: 74104). On-column DNA digestion was performed using RNase-Free DNase Set (Qiagen:79254). DNase I stock solution was prepared by dissolving lyophilized DNase I (1500 Kunitz units) in 550 µL of RNAse-free water. All centrifugation for spin columns was carried out at 16,000 g. Cells preserved in RNAlater were pelleted by centrifugation at 500 g for 5 minutes. If the pellet is loose, an additional brief centrifugation was performed at up to 7000 rpm for 5 seconds using a SCILOGEX D1008 Mini Centrifuge. The cell pellet was then resuspended in 600 µL of RLT Buffer with 1% 2-mercaptoethanol (6 µL 2-mercaptoethanol per 600 µL RLT Buffer). Cell lysates were homogenized by passing through a 20-gauge needle fitted to an RNAse-free syringe at least 10 times. One volume (600 µL) of 70% ethanol was added to the homogenized lysates and mixed by pipetting. Each sample was transferred to a RNeasy spin column and centrifuged for 30 seconds. Then, 350 µL of Buffer RW1 was added to each column, which was then centrifuged for 30 seconds. DNase I working solution was prepared by mixing the DNase I stock solution with Buffer RDD 1:7 (add 70 µL Buffer RDD to 10 µL of DNase I). To perform on-column DNase digestion, 80 µL of DNase I working solution was added directly to the center of the column membrane, followed by 15 minutes of incubation at room temperature. After incubation, the DNase I was washed away by adding 350 µL of Buffer RW1 and centrifuging for 30 seconds. Two consecutive washes with 500 µL of Buffer RPE were then performed. For the first wash, the samples were centrifuged for 30 seconds, whereas for the second wash, they were centrifuged for 2 minutes. An final 1-minute centrifugation in a fresh collection tube was performed to remove residual wash buffer. The spin columns were then transferred to a fresh 1.5 mL tube for elution. For all groups, 30 μL RNAse-free water was added and incubated for 2 minutes to elute the RNA. RNA was then collected by centrifugation for 1 minute. Concentrations (ng/µL) and quality (A280/A260, A230/A260) of the RNA samples were measured by a Nanodrop™ One Spectrophotometer (Thermo Fisher Scientific: ND-ONE-W). RNA samples were stored at -80 °C and thawed only right before reverse transcription. To convert the reporter transcripts into cDNA, reverse transcription was performed using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific: 18091050). This kit allows gene-specific, customized primers. For HCR-seq, the primer used in reverse transcription is called the RT_UMI_primer, which binds specifically to the reporter transcripts and adds to the cDNA a 15-bp random sequence that serves as the unique molecular identifier (UMI; also referred to as the transcript barcode). The sequence of the RT_UMI_primer can be found in Supplementary Table 4). The design of the RT_UMI_primer is specific for reporter transcripts and allows barcoding of each first-strand cDNA. This primer also adds part of the Illumina adapters. Later, during data analysis, the transcript barcodes can be used to adjust for amplification bias and as a means of correcting for sequencing errors. The reverse transcription reaction was set up according to the following procedure. First, the annealing reaction mix (13 µL) was set up by combining 4 µL of total RNA, 1 µL of dNTP mix (10 mM), 1 µL of RT_UMI_primer (2 µM), and 7 µL of DEPC-treated water. Primers were annealed to the RNA by heating the annealing reaction mix at 65 °C for 5 minutes and incubating on ice for at least 1 minute. Next, the reverse transcription reaction mix (7 µL) was prepared by combining 4 µL of SSIV Buffer (5X), 1 µL of DTT (100 mM), 1 µL of ribonuclease Inhibitor, and 1 µL of SuperScript IV Reverse Transcriptase (200 units/µL). The reverse transcription reaction mix and the annealing reaction mix were then combined. The reaction mixture (20 µL) was incubated at 50 °C for 10 minutes and subsequently inactivated by incubation at 80 °C for 10 minutes. To remove RNA, 1 µL of RNase H was added, followed by incubation at 37 °C for 20 minutes. The first-strand cDNA was then cleaned up twice using the Monarch Spin PCR & DNA Cleanup Kit (5 ug) (New England Biolabs: T1130S). A second cleanup was required because a single cleanup could not reduce the amount of residual RT_UMI_primer to a level low enough so that the primer wouldn't interfere with the downstream PCR processes. In the first cleanup, the cDNA was eluted with 50 µL of Elution Buffer, whereas in the second cleanup, the cDNA was eluted with 12 µL of Elution Buffer.