GSM1463994: Input; Mus musculus; OTHER
Genomic DNA from C57bl6 mouse thymus was sheared by sonication (Diagenode pico) and DNA fragments between 330 bp and 430 bp were size-selected on a 1.2% high resolution agarose gel (Sigma-Aldrich) and purified using Qiaquick gel extraction kit (Qiagen). Ion Torrent adapters were ligated to 8 µg of size-selected DNA fragments using Ion Plus Fragment Library Kit (Life Technologies) following manufacturer’s instructions except for the final PCR amplification. Forty PCR reactions (98°C for 2 minutes; followed by ten cycles of 98°C for 20s, 65°C for 15s, 72°C for 15s) with 2µL of adaptor ligated DNA were performed using KAPA Hifi Hot Start Ready Mix (KAPA Biosystems) and primers (fw: TAGAGCATGCACCGGAACCATCTCATCCCTG*C*G*T*G and rw: GGCCGAATTCGTCGAAACCACTACGCCTCCGC*T*T*T*C, * indicates phosphorothioates bonds) which add a specific 15 nucleotides extension to both adapters for directional cloning using recombination (In Fusion HD, Clontech). The forty PCR reactions were pooled and purified with Agencourt AMPureXP DNA beads (Ratio beads/PCR 1.2; Beckman Coulter). For target enrichment, a home-designed oligonucleotide microarray covering the 7,542 CRMs defined above was constructed using the SureSelect technology (Agilent) and the eArray tool (https://earray.chem.agilent.com/earray/). The genomic library was hybridized on the microarray as outlined in the Agilent SureSelect DNA Capture Array protocol (Agilent). Briefly, an hybridization mixture with 10 μg of PCR along with Blocking oligo 1 (TAGAGCATGCACCGGAACCATCTCATCCCTG*C*G*T*G), Blocking oligo 2 (GGCCGAATTCGTCGAAACCACTACGCCTCCGC*T*T*T*C), Blocking oligo 3 (CACGCAGGGATGAGATGGTTCCGGTGCATGC*T*C*T*A), Blocking oligo 4 (GAAAGCGGAGGCGTAGTGGTTTCGACGAATTC*G*G*C*C), was denatured at 95°C for 3 minutes followed by transfer to 37°C for 30 minutes. The hybridization mixture was dispensed onto the center of a gasket slide and the oligonucleotide array was placed with the active side contacting the hybridization mixture. The resulting sandwich was incubated at 65°C for 65 hours on a rotisserie in an Agilent hybridization oven. Following hybridization, the slide-gasket sandwich was disassembled and the µarray was washed, following manufacturer’s instructions. Slides were dried and 490 μL of nuclease-free water were added to a new gasket slide and the dried µarray was placed atop as described earlier, creating a fresh slide-gasket sandwich. The slide-gasket sandwich was incubated at 95°C 10 minutes on a rotisserie in an Agilent hybridization oven. After heat denaturation, the eluate was recovered with a 1-ml 30G syringe. The eluted DNA was purified with Agencourt AMPureXP DNA beads (Ratio beads/PCR 1.8) and eluted in 80 µL. Twenty independent 25 µL PCR (98°C for 2 minutes; followed by 20 cycles of 98°C for 20s, 65°C for 15s, 72°C for 15s) reactions with 4 µL of captured DNA were performed using KAPA Hifi Hot Start Ready Mix and primers (fw: TAGAGCATGCACCGGAACCATCTCATCCCTG*C*G*T*G and rw: GGCCGAATTCGTCGAAACCACTACGCCTCCGC*T*T*T*C). PCR reactions were pooled, purified with Agencourt AMPureXP DNA beads (Ratio beads/PCR 1.8) and eluted in 200 µL H2O. The mammalian STARR-Seq screening vector has been previously described. The screening vector was linearized by a 6 hours digestion with AgeI-HF and SalI-HF (New England Biolabs), followed by agarose gel electrophoresis, QIAquick gel extraction (Qiagen) and a clean-up with Qiagen Minelute PCR purification Kit (Qiagen). The purified PCR products were recombined (Clontech In-Fusion HD) with the screening vector (Qiagen) in a total of ten 10 µL reactions. The recombination reactions were pooled, purified with Agencourt AMPureXP DNA beads and eluted in 29 µL. Thirteen aliquots (20 µL each) of MegaX DH10B Electrocompetent Bacteria (Life technologies) were transformed with 2 µL of DNA each, according to the manufacturer’s recommendation. After one hour recovery at 37°C, transformations were pooled and transferred to 2 L of LBAMP medium and grown overnight. An aliquot of the transformation was plated on LBAMP to estimate the number of cloned fragments. Finally, the plasmid library was extracted using Qiagen Plasmid Plus Mega Kit (Qiagen). Transfection of library (5µg/1x10^6 cells) was performed using Neon Transfection System (Life technologies). A total of 30x10^6 cells were transfected per experiment. 25x10^6 cells were used for RNA purification and 5x10^6 for DNA purification. P5424 cells were transfected with 1600v-20ms-1 pulse and 3T3 with 1400v-20ms-2 pulses. Cells were transferred to complete growth medium and incubated for 24 hours before RNA and DNA isolation. Total RNA 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). Plasmid DNA was extracted from 5x10^6 cells using a QIAGEN plasmid plus mini kit (Qiagen), following manufacturer’s instructions. 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. After reverse transcription, 1 µL of RNaseH was added (37°C, 1h). cDNA was purified with QIAquick PCR purification kit and concentration was determined with Qubit ssDNA Kit (Life technologies). 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. To remove any residual contamination of plasmid or cDNA, PCR products were purified on gel using QIAquick gel extraction Kit (Qiagen) followed by a clean-up with QIAquick minelute PCR purification kit (Qiagen). To generate the Ion Torrent libraries, the purified PCR was used as template for the second PCR (5 ng/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 the KAPA Hifi Hot Start Ready Mix and Ion Torrent library amplification primer mix (Life technologies, T_PCR_A: CCA TCT CAT CCC TGC GTG TC and P1amp: CCA CTA CGC CTC CGC TTT CCT CTC TAT G). 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). To assess change in library composition caused by electroporation, 10 reactions with 5 ng per reaction of reporter constructs isolated from transfected cells were amplified as described above. The emulsion PCRs were performed with 25 µL of Starr-Seq libraries (10 pM) using the Ion PGM Template OT2 400 kit and the ONE TOUCH 2 system (Life Technologies), according to manufacturer’s instructions. Template-positive Ion Sphere Particles were enriched using the Ion PGM Enrichment Beads (Life Technologies), according to manufacturer’s instructions.