title: GSM2615956: HeLa_BR_3_TR_1_Nuclei; Homo sapiens; OTHER
library_construction_protocol: RNA was isolated by TRIzol (ThermoScientific) - chloroform extraction, followed by isopropanol precipitation, according to standard procedures. 2 µg of BioAnalyzer-validated RNA were digested with recombinant DNase-I (2.77 U/µl, Worthington #LS006353) at 37°C for 30 min, followed by heat-inactivation at 75°C for 10 min. Reverse transcription was performed with SuperScript III cDNA synthesis kit (ThermoScientific). Quantitative RT-PCR was performed using the FastStart Universal SYBR Green Master mix (Roche) on an ABI 7900. Primers were: NEAT1 forward TGATGCCACAACGCAGATTG, reverse GCAAACAGGTGGGTAGGTGA, and SNHG5 forward GTGGACGAGTAGCCAGTGAA, reverse GCCTCTATCAATGGGCAGACA. After processing the raw data by qPCR Miner (PMID 16241897), the efficiency of each primer set was used to calculate the relative initial concentration of each gene. The relative expression in the nuclear and cytoplasmic fractions was then calculated by normalization to that in the whole cell. Sequencing libraries were prepared by PCR amplification using PfuUltra II Fusion DNA polymerase (Agilent #600672) and primers designed to anneal to the universal primer site flanking the oligos and to add sequencing index barcode for multiplexing: forward caagcagaagacggcatacgagatCGTGATgtgactggagttcagacgtgtgctcttccgatctACTGGCCGCTTCACTG, reverse AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT (capital letters indicate (1) the index for the library and (2) the region complementary to the universal primer site). PCR amplification (initial denaturation 95°C – 2 min; cycling 95°C – 30 secs, 55°C – 30 secs, 72°C – 30 sec; final extension 72°C – 10 min) was carried out for 30 cycles followed by triple 0.6x, 1.6x, and 1x SPRI beads (Agencourt AMPure XP, Beckman Coulter) cleanup. The quality and molarity of the libraries was evaluated by BioAnalyzer and the samples were sequenced in a pool of 6 on the Illumina HiSeq2500, full flow cell, single-read 100 bp.