GSM5133578: KO-TRAM-PBS-1_RNA; Mus musculus; RNA-Seq
RNA was extracted into a 30-µL volume using the RNeasy Mini Kit (74104, Qiagen) and RNase-Free DNase Set (79254, Qiagen), following the manufacturer's instruction. 10,000 cells were used to produce each RNA-seq library, with two replicates for each genotype and experimental condition. The extracted mRNA was then concentrated by ethanol precipitation and resuspended in 4.6 µL of RNase-free water. Next, we used the SMART-seq2 protocol, with minor modifications, to prepare cDNA. 2 µL of oligo-dT primer (100 µM) and 2 µL of dNTP mix (10 mM) were added to 2 ng of mRNA in 4.6 µL of water.  The mRNA solution was denatured at 72 °C for 3 min, then immediately placed on ice. Next, 11.4 µL of reverse transcription mix [1 µL of SuperScript II reverse transcriptase (200 U/µL), 0.5 µL of RNAse inhibitor (40 U/µL), 4 µL of Superscript II first-strand buffer, 1 µL of DTT (100mM), 4 µL of 5 M Betaine, 0.12 µL of 1 M MgCl2, 0.2 µL of TSO (100 M), 0.58 µL of nuclease-free water] was added to the mRNA solution. For the reverse transcription reaction, the solution was incubated at 42 °C for 90 min, followed by 10 cycles of 50 °C for 2 min, 42 °C for 2 min, then inactivation at 70 °C for 15 min. 20 µL of the resulting solution (first-strand mixture) was then mixed with 30 µL of PCR mix [25 µL KAPA HiFi HotStart ReadyMix, 0.5 µL IS PCR primers (10 µM), 0.5 µL Evagreen dye, and 4 µL nuclease-free water] and amplified using the program 98 °C for 1 min, followed by 9-11 cycles of 98 °C 15 s, 67 °C 30 s, 72 °C 6 min. Finally, the cDNA was purified using 50 µL of SPRIselect beads. RNA-seq libraries were generated with the Nextera XT DNA Library Preparation kit (FC-131-1024, Illumina) and manufacturer's protocol, using approximately 600 pg of purified cDNA from each sample.