title: "human nanoCAGE libraries prepared with a serial dillution of Hep G2 total RNA: 1250, 250, 50 or 10 ng."
design: "After extraction of total RNA with commercial kits, it was necessary to increase its concentration. The volume of water was reduced to 2 microl by centrifugal evaporation at room temperature in the presence of 0.66 M D- Threalose (Nacalai Tesque), 3.3 M D-Sorbitol (WAKO), 100 microM of TS oligonucleotide TAGTCGAACTGAAGGTCTCCAGCA(rG)(rG)(rG), 10 microM of random RT primer GTACCAGCAGTAGTCGAACTGAAGGTCTCCTCTN15 (Stangegaard et al., 2006) or 10 microM of polythimine RT primer GTACCAGCAGTAGTCGAACTGAAGGTCTCCTCT18. The oligonucleotides were added at this step because it helps to keep the final reaction volume low. They contain binding sites for the type III restriction enzyme EcoP15I (Meisel et al., 1992), indicated in bold, that will be used to extract 5' tags from the cDNAs. Trehalose and sorbitol protect RNAs against degradation and capture on tube walls by preventing the samples from drying completely. Furthermore, these poly-hydroxylated molecules thermoactivate the reverse transcriptase (RTase) at the next step (Carninci et al., 1998). The mixture was then was heat-denatured at 65 deg_C for 10 min in a thermocycler (MWG) and transferred quickly on a ice/water mixture, to avoid the formation  of  secondary  structures.  Reverse transcription and template switching were accomplished together in a volume of 10 microl with the following components: 1.25 x first strand buffer (Invitrogen), 650 nM dNTPs (TaKaRa), 1.3 mM DTT (Invitrogen), 925 mM betaine (Spiess and Ivell, 2002) (WAKO) , and 200 units of SuperScriptII (Invitrogen). The reactions were incubated at 22 deg_C for 10 min, 50 deg_C for 30 min, 75 deg_C for 15 min in a thermocycler., and then immediately transferred on a ice/water mix. To prepare the second strand synthesis and amplification of the cDNAs, a small scale semi-suppressive PCR reaction was performed to evaluate the optimal number of cycles, defined as the last cycle before the intensity of the product ceases to increase. 10 microl aliquots were taken every two cycles and analyzed on 1% agarose gel. For each cDNA preparation, 2 microl of first strand cDNA were amplified in reactions of 100 microl using a mixture containing 1 x ExTaq Buffer (TaKaRa), 200 microM dNTPs (TaKaRa), 100 nM forward PCR primer TAGTCGAACTGAAGGTCTCCAGC, 100 nM reverse PCR primer primer GTACCAGCAGTAGTCGAACTGAAGGTCTCCTCT, and 5 units of ExTaq (TaKaRa) with the following thermocycling program: 5 min 95 deg_C, n x (10 s at 95 deg_C, 15 s at 65 deg_C, 2 min at 68 deg_C) and using hot start. The rest of the cDNAs (6 microl each) were then processed in 4 reactions of 100 microl with the chosen number of cycles. PCR products were pooled and cleaned using CTAB and GE Healthcare GFX purification columns (to be published elsewhere). Tags were prepared from 45 microl of cDNA by enzymatic cleavage in 100 microl using 100 units of EcoP15I (NEB), 1 x buffer 3 (NEB), 1 mM ATP (NEB) and 100 ng/microl BSA (NEB), at 37 deg_C for 4 hours. We chose EcoP15I (Meisel et al., 1992) for tag generation, as it was reported to cut 25/27 bases downstream its binding site (Matsumura et al., 2003). In addition its non-palindromic DNA binding site strongly reduces the opportunities for the formation of PCR primer dimers during subsequent steps. Reactions were mixed with 130 microl of TE buffer and the low molecular weight cleavage products were purified through the Microcon YM-100 membranes (Millipore) by spinning 15 min at 500 x g. The columns were filled again with 130 microl of TE buffer and spun 15 min at 500 x g. The flow-through was then concentrated on Microcon YM-10 (Millipore) by spinning 20 min at 12,000 x g. The low molecular weight cleavage products were recovered by spinning 3 min at 1000 x g after flipping the cartridges. Ligation adapters were annealed by mixing equimolar amounts of two oligonucleotides, NNAGCTGTAGAACTCTGAACCTGT and ACAGGTTCAGAGTTCTACAGCT, in a thermocycler at 95 deg_C and left to cool down to room temperature. 1 pmol of adapters was ligated to 10 microl of the EcoP15I cleavage products in 20 microl of 0.5 x "Mighty" DNA ligation mixture (TaKaRa) overnight at 16 deg_C in a thermocycler with lid heater (MWG). Optimal number of cycles for the ligation product to be amplified was determined  by  PCR  with  5 microM  of  forward  PCR  primer AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAG, 5 microM of  reverse  PCR  primer CAAGCAGAAGACGGCATACGATAGTCGAACTGAAGGTCTCCAG, 1 x ExTaq buffer (TaKaRa), 200 microM dNTPs (TaKaRa), 5 units of ExTaq (TaKaRa). The program was: 5 min 95 deg_C, n x (10 s at 95 deg_C, 10 s at 68 deg_C). 3 PCR reactions in 100 microl for each sample were performed. The excess of the primers was digested with 5 units of Exonuclease I (TaKaRa) at 37 deg_C for 30 min and then the enzyme was heat inactivated at 55 deg_C for 15 min. Then the PCR products were purified by electrophoresis on 8% polyacrylamide gel and the band corresponding to the expected size (114 bp) was cut, passed through a syringe to break the structure of polyacrylamide, and the DNA was extracted at room temperature in a microtube on rotation with 800 microl of 1 x TE buffer over night. The tubes were centrifuged at 13,000 rpm for 10 min and the supernatant was collected. 600 microl of 1 x TE buffer were added to the polyacrylamide in the microtube and let to rotate at room temperature for 1 hour. This step was repeated once more. Then for each sample, all the collected fractions were pooled together and passed through a Microspin filter (GE Healthcare) to eliminate residual traces of polyacrylamide. A total of 2 ml of filtrate was concentrated to 100 microl passing through a Microcon YM-10 column (Millipore). The purity and the concentration of the sample was estimated by Nanodrop UV spectrophotometer and Agilent 2100 Bioanalyzer and an aliquot of 10 microl was analyzed by polyacrylamide gel electrophoresis to check for the correct size of the recovered DNA."