GSM2198559: CapStarr-seq HeLa replicate 1; Homo sapiens; OTHER
Total RNA CapStarr-seq transfected library from given cell line was extracted using RNeasy miniprep kit (Qiagen) with the on-column DNaseI treatment. The PolyA RNA fraction was isolated using µMACS mRNA isolation kit (Miltenyi) following manufacturer’s instructions. PolyA RNA was treated with Ambion turboDNase (Ambion) and purified with RNeasy Minelute kit (Qiagen). Finally, mRNA was quantified with Qubit RNA HS Kit (Life technologies). 1st strand cDNA synthesis was performed with superscript III (Life technologies) using a reporter-RNA specific primer (CAAACTCATCAATGTATCTTATCATG) and 0.2 to 0.3 µg of polyA RNA per reaction for a total of 10 reactions. cDNA was purified with QIAquick PCR purification kit and concentration was determined with Qubit ssDNA Kit (Life Technologies). Libraries were prepared follow NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB, E6000S). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo-) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 7 cycles and library fragments of ~400 bp (insert plus adaptors and PCR primer sequences) were cleaned up using AMPure XP beads (ratio bead/PCR = 1.2). Libraries were then processed follow CapStarr-seq procedure. Total RNA was isolated as described above. cDNA was amplified by a 2-step nested PCR using the KAPA Hifi Hot Start Ready Mix. In the first PCR (98°C for 2 minutes; followed by 15 cycles of 98°C for 20s, 65°C for 20s, 72°C for 30s), 5 ng of cDNA per reaction were amplified using 2 reporter-specific primers (fw: GGGCCAGCTGTTGGGGTG*T*C*C*A*C and rw: CTTATCATGTCTGCTCGA*A*G*C), one of which spans the splice junction of the synthetic intron, in a total of 10 reactions. The PCR product was then clean-up with QIAquick minelute PCR purification kit (Qiagen). To generate the Illumina libraries, the purified PCR product was used as template for the second PCR (2ng/PCR, for a total of 10 PCR; 98°C for 2 minutes; followed by 10 cycles of 98°C for 20s, 65°C for 20s, 72°C for 30s), with Universal primer and Index Primer (Illumina) using the KAPA Hifi Hot Start Ready Mix. To generate the INPUT control, ten reactions with 5 ng of reporter constructs (library) per reaction were amplified using the same conditions as above, at the exception of the forward primer in the 1st PCR (fw: GGGCCAGCTGTTGGGGTG*A*G*T*A*C). The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. Libraries were run on Illumina NextSeq500.