GSM7872971: NE-RNA-2; Homo sapiens; RNA-Seq
Cell preparation: Cell culture medium was removed by centrifuging at 300g for 5 minutes at 4°C. Cells were washed twice with 1 mL cold PBS and suspended in 60 μL PBS supplemented with 0.6 μL PIC and 0.6 μL PMSF solution. Cells were then stored on ice until used for ChIP-seq or RNA-seq. RNA-seq library construction: Each RNA-seq library was prepared using 25,000 cells. We prepared two technical replicates for each condition. Total RNA was extracted using the RNeasy Mini Kit (74104, Qiagen) and RNase-Free DNase Set (79254, Qiagen). The extracted total RNA was suspended in 30 μL RNase-free water and concentrated by ethanol precipitation. Total RNA was resuspended in 4.6 μL of RNase-free water supplemented with 5% RNase inhibitor (40 U/μL). SMART-seq2 protocol (Picelli et al., Nature Protocols 9 (2014) 171–181.) with minor alterations (Zhu et al., Cell Reports 37 (2021) 109836, Naler et al., Commun Biol 5 (2022) ) was then used for reverse transcription to produce cDNA. 2 μL of oligo-dT primer (10 μM), 2 μL of dNTP mix (10 mM) and 4.3 μL of total RNA solution were first mixed well. This mixture (8.3 μL) was denatured at 72°C for 3 minutes and immediately stored on ice. Next, 11.7 μL of reverse transcription mix (1μL of SuperScript II reverse transcriptase (200 U/μL), 0.5μL of RNase inhibitor (40 U/μl), 4μL of Superscript II first-strand buffer, 1 μL of DTT (100 mM), 4 μL of 5 M Betaine, 0.12 μL of 1 M MgCl2, 0.2 μL of TSO (100 μM), 0.88 μL of nuclease-free water) was added to the 8.3μl mixture. The total reverse transcription reaction mix was incubated in a thermal cycler at 42°C for 90 minutes, followed by 10 cycles of (50°C for 2 minutes and 42°C for 2 minutes), and finally inactivated at 70°C for 15 minutes. The reverse transcription reaction mix was then mixed with 30 μL of PCR mix (25 μL KAPA HiFi HotStart ReadyMix, 0.5μL IS PCR primers (10 μM), 2.5μL Evagreen dye, and 2μL nuclease-free water) and amplified with the program: 98°C for 1 minute, 9–11 cycles of (98 °C for 15 seconds, 67 °C for 30 seconds, 72 °C for 6 minutes). Finally, the cDNA was purified using 40 μL of SPRIselect beads and resuspended in ~7 μL of low-EDTA TE buffer.   The RNA-seq library was made using Tn5-tagmentation. Tn5 transposase (14-17 mg/ml) was produced following a published protocol (Picelli et al., Genome Res 24 (2014) 2033–2040.). Tn5 transposase and T5/T7 transposons were assembled to form Tn5 transposome using a published protocol (Amini et al., Nat. Genet. 46 (2014) 1343-1349). Briefly, 1.5 μl of 100 μM T5 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS (a 5'-phosphorylated 19-bp mosaic end complementary oligonucleotide, IDT), while 1.5 μl of 100 μM T7 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS. 3 μl of T5-pMENTS solution and 3 μl of T7-pMENTS solution were separately incubated at 95°C for 5 min followed by a slow ramp to 25°C at -0.1°C/sec. 2.5 μl of T5-pMENTS solution was then mixed with 2.5 μl of Tn5 transposase (14-17 mg/ml), while 2.5 μl of T7-pMENTS solution was mixed with 2.5 μl of Tn5 transposase. T5-Tn5 solution and T7-Tn5 solution were separately incubated at 37°C for 1 h to form T5-Tn5 transposome and T7-Tn5 transposome, respectively. 4 μl of T5-Tn5 transposome and 4 μl of T7-Tn5 transposome were mixed to form 8 μl of assembled Tn5 transposome before it was mixed with 1 μl cDNA (containing ~500 pg cDNA) and 1 μl reaction buffer (TNP92110, Lucigen). The reaction mixture was incubated at 37 °C for 1 h. 1 μl of 10× stop solution (TNP92110, Lucigen) was added to the reaction mixture to stop tagmentation. Two rounds of 1× SPRIselect beads cleanup were performed to purify tagmented cDNA. The tagmented cDNA was then eluted in 10 μl low EDTA TE buffer. 25 μl KAPA HiFi HotStart ReadyMix was activated at 98 °C for 30 sec and then mixed with 9.5 μl tagmented cDNA and 10 μl Nuclease-free water, followed by incubation at 72 °C for 5 min. 1.5 μl of 25 μM P5 primer, 1.5 μl of 25 μM P7 primer and 2.5 μl 20x Evagreen were then added. The following amplification program was used to achieve a 300-400 RFU increase: 98 °C for 30 s; 11-12 cycles of (98 °C for 10 seconds, 63 °C for 30 seconds, 72 °C for 30 seconds) and 72 °C for 1 minute. Amplified library was purified with 0.8× SPRIselect beads and eluted in 8 μl low EDTA TE buffer. Libraries were sequenced with Illumina HiSeq 4000 with single-end 50 nt read length.