title: GSM2460275: PSC 100 cells narrow CP100N; Homo sapiens; RNA-Seq
library_construction_protocol: The cells were either diluted into phosphate buffer saline (PBS) with 0.1% polyvinyl alcohol (PVA) before lysis or sorted directly into a lysis buffer . After incubation at 37°C for 30 minutes and inactivating proteinase K, the lysate was polyadenylated in a final 4 µl of 1X PAP mixture (37°C for 15 minutes, 45°C for 5 minutes, 55°C for 5 minutes, and 65°C for 20 minutes). After supplementing 0.5 µl of 20 µM i7T20V oligo, the reaction was heated at 75°C for 1 minute and cooled back to 42°C. 5.5 µl of RT mixture were added to final 1X, and then the mixture was incubated at 42°C for 90 minutes and 70°C for 15 minutes. If further preamplification was required, the cDNA was added to 40 µl of preamplification mixture. The thermal cycling were performed with initial denaturation at 98°C for 1 minute followed by cycles at 98°C for 10 seconds, 65°C for 15 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes.   The cDNA from RT reaction/preamplification, or eluted DNA from size selection was concentrated with carboxylated beads and PEG/NaCl (Rohland and Reich, 2012). Briefly, 1 µl of magnetic beads and 3 volumes of PEG/NaCl solution were mixed with the solution containing DNA. The mixtures were left at ambient temperature for 15 minutes. After being washed two times with freshly prepared 80% ethanol, the beads were dried at ambient temperature for 15 minutes on a magnetic stand and eluted with 0.1% Triton X-100 for another 15 minutes at ambient temperature.  After preamplification and concentration with PEG/NaCl, the cDNA was run in 6% denaturing PAGE and stained in a 1X TBE buffer containing 1X SYBR GOLD for 20 minutes. After visualization and size-selection, the gel pieces were crushed by spun through a 0.5 ml microtube with a pinhole at the bottom and soaked in 300 µl of 10 mM Tris 8.0, 300 mM NaCl, and 1 mM EDTA at ambient temperature overnight. After concentration with PEG/NaCl, the eluted cDNA was appended with P5 and P7 adapters by 10 cycles of PCR with KAPAHiFi DNA polymerase (conditions identical to preamplification, except P5PCRIIA and N7xx were used as primers). After another round of PEG/NaCl to remove primers, equal amounts of the indexed libraries were mixed for sequencing with the Illumina Hiseq 2500 system.
attributes.tag: GEO Accession
attributes.value: GSM2460275