GSM7393177: defined_end_tcell biol rep 2-1; Homo sapiens; RNA-Seq
Cells were lysed in the presence of cycloheximide to halt translating ribosomes so that polysome profiling could be run with the lysate. Lysis and RNA isolation for HEK293T and HepG2 were performed as described in Sample, Wang et al. 2019. For T cells, cycloheximide was first added dropwise to the media to a final concentration of 100 ug/mL and incubated for 5 additional minutes. Cells were then spined down at 1500 rpm for 5 min and the supernatant was discarded. 300 uL of cold lysis buffer (Sample, Wang et al. 2019) was used to resuspend the cells, and the mixture was incubated on ice for 10 minutes. Cells were then triturated by passing the mixture through a 25-gauge needle ten times. The mixture was spined down at 16,000 rpm for 5 min at 4C and the supernatant was transferred to another tube. 1.5 uL of 1U/uL DNAse was added, the mixture was set on ice for 30 minutes, and stored at -80C. Polysome profiling was performed as in Sample, Wang et al. 2019. Sequencing libraries for the defined-end reporters were constructed for each extracted polysome fraction as in Sample, Wang, et al. 2019. For the random-end reporter libraries, sequencing library preparation comprised reverse transcription, template switching, and qPCR amplification, all of which were performed separately for each polysome fraction. For reverse transcription, we first mixed 10.5 uL of purified RNA with 2 uL of 2 uM RT primer (AGGGACATCGTAGAGAGTCGTACTTANNNNNNNNNNAGATGAACTTCAGGGTCAGC, where Ns comprise the UMI) and 2uL of 10mM dNTP mixture (NEB), incubated the mixture at 65C for 5 minutes, and placed on ice for at least one minute. Then, we added 4 uL Maxima RT buffer, 0.5 uL Superase-In RNAse inhibitor (Thermo Fisher), and 1 uL Maxima RT RNaseH minus Enzyme (Thermo Fisher), incubated at 50C for 15 min, then 85C for 10 min, and transferred back to ice. Next, we added 1 uL RNase (from bovine pancreas, DNase free, Roche) and 1 uL RNase H (NEB) and incubated at 37C for 15 minutes. Finally, the product was cleaned with KAPA beads (Roche) with a 3x ratio of beads to DNA volume, and resuspended in 25 uL dH2O. For template switching, we added 8 uL Maxima RT buffer, 6 uL 50% PEG-8000, 0.5 uL Superase-In, 1 uL Maxima RT RNaseH minus enzyme, and 0.5 uL of 10 uM template switching oligo (AAGCAGTGGTATCAACGCAGAGTACATrGrGrG). This mixture was incubated at 42C for 30 minutes, then 85C for 10 minutes. Next, 1uL RNase (Roche) and 1uL RNase H (NEB) were added and the mixture was incubated at 37C for 15 minutes. Finally, the product was cleaned with 3x KAPA beads and resuspended in 20 uL dH2O.  qPCR amplification was performed using the KAPA HiFi master mix (Roche) with forward primers AATGATACGGCGACCACCGAGATCTACAC[8nt-long index barcode]AGCGTGACAGGGACATCGTAGAGAGTCGTACTTA and reverse primers CAAGCAGAAGACGGCATACGAGAT[8nt-long index barcode]AAGCAGTGGTATCAACGCAGA, where the barcodes were specific to each polysome fraction. qPCR reactions were stopped before reaching saturation and purified via gel extractions.