GSM2342976: Patient 04 Normal; Homo sapiens; OTHER
Total RNA was isolated using the miRNeasy Kit (Qiagen) according to manufacturer instructions. We performed PAS-seq with extensive modifications. Total RNA was fragmented and DNase treated by incubating with 1.5X First Strand Buffer (ThermoFisher Scientific) and 50U/mL RQ1 RNase-free DNase (Promega) for 30 minutes at 37C, followed by 3 minutes at 95C. The RNA was then concentrated using the RNeasy minElute kit (Qiagen), and the concentrated, fragmented RNA was reverse transcribed using SuperScript II (ThermoFisher Scientific) as follows: First, 1µg of RNA was mixed with 1µL each of primers 1320 UMI-DT2 (TVN primer with UMI) and 1290 SMART-TRUSEQ (template switching primer) in 12.5µL total volume and incubated at 65C for 5 minutes, followed by a hold at 42C. Next, 4 µL 5X First-strand buffer, 2 µL 100 mM DTT, 0.5 µL RNasin Plus (Promega), and 1 µL SuperScript II were added to the RNA+primers (all pre-warmed to 42C). The reaction was held at 42C for 50 minutes, followed by 15 minutes at 70C. The resulting cDNA was then amplified using Phusion Hot Start II (ThermoFisher Scientific) as follows: 4µL of cDNA and 1µL of TRU-SEQ barcoded primer mix (Illumina) were used in a standard 25µL reaction with the following program: [1. 98C 2:00,  2. 98C 0:10, 3. 60C 0:30, 4. 72C 0:30, 5. repeat steps 2-4 2x, 6. 98C 0:10, 7. 72C 1:00, 8. repeat steps 6-7 24x, 9. 72C 10:00]. Resulting PCR products were then run on a 2% SeaPlaque agarose (Lonza) gel. The region between 250 and 380bp was excised and the DNA purified using the minElute Gel Kit (QIagen). The resulting libraries were then sequenced on an Illumina Hiseq 2000 (50bp, single end).
GEO Accession
GSM2342976