title: GSM2545195: SKBR3 rep3 (GRO-Seq); Homo sapiens; OTHER
library_construction_protocol: Nuclei Preparation.  All cells were grown using the growth conditions listed above at the LONESTAR Consortium cell culture core facility.  The cells were collected at ~70-80% confluence.  Nuclei were isolated from fresh (not frozen) cells, as described previously (Luo et al., 2014).  Briefly, the cells were washed three times with ice-cold PBS and swollen in Hypotonic Lysis Buffer [10 mM Tris•HCl pH 7.4, 0.5% NP-40, 10% glycerol, 3 mM CaCl2, 2 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail (Sigma-Aldrich), and SUPERase-In (Ambion)].  The swollen cells were collected by centrifugation at 500 x g for 10 min at 4°C.  The cell pellets were resuspended in 1.5 ml of Hypotonic Lysis Buffer and pipetted up and down through a narrow opening 30 to 50 times to release the nuclei.  The nuclei were collected by centrifugation and washed once with 1 mL of ice cold Lysis Buffer.  The nuclei were collected by again centrifugation and washed once with 1 mL of Hypotonic Lysis Buffer.  After a final collection by centrifugation, the resulting pelleted nuclei were resuspended in 500 μL of Freezing Buffer (50 mM Tris•HCl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 4 units/mL of SUPERase-In per mL), counted, frozen in liquid nitrogen in 100 μL aliquots containing 5 x 106 nuclei, and stored at -80°C until use. Nuclear run-on and GRO-seq library preparation were performed as previously described (Hah et al., 2011), with modifications (Danko et al., 2013; Luo et al., 2014).  Libraries were prepared from two biological replicates using a circularized ligation-based protocol for adaptor addition for improved efficiency of library preparation, reduced sequence bias, and ease for barcoding.  Briefly, nuclear run-ons were extended for ~100 bases in the presence of Sarksoyl (to prevent reengagement of RNA polymerases), rNTPs, α32P-CTP, and 5-bromo-UTP.  The nascent RNAs were isolated, hydrolyzed to ~150 bases, and enriched using α-BrdUTP antibody-conjugated agarose beads (Santa Cruz).  Enriched run-on RNA was subjected to polyA tailing with E. coli PolyA Polymerase and subsequently converted to cDNA by reverse transcription using SuperScript III reverse transcriptase (Invitrogen) and an RT primer (pGATCGTCGGACTGTAGAACTCT/idSp/CCTTGGCACCCGAGAATTCCATTTTTTTTTTTTTTTTTTTTVN), where p indicates phosphorylation, idSp indicates dSpacer Furan, and VN indicates degenerate nucleotides.  Reverse transcribed cDNAs were size selected (120 to 400 bp) by denaturing PAGE and circularized with CircLigase (Epicentre) to position Illumina adapter sequences relative to the 5’ and 3’ ends of reverse-transcribed run-on cDNA.  The circularized cDNAs were re-linearized by with ApeI (NEB), purified by phenol-chloroform extraction, and PCR amplified using unique Illumina TrueSeq small RNA sample barcoded primers with Phusion high fidelity DNA polymerase (NEB).  The PCR products (175 bp to 300 bp) were separated by native PAGE, eluted, and purified.  After library quality control assessment using a Bioanalyzer (Agilent), the samples were subjected to 50 bp single-end sequencing using an Illumina HiSeq 2000 Sequencing System.  At least two biological replicates for each cell line were sequenced