GBS libraries were constructed using the two-enzyme modification of the original GBS protocol (Elshire et al. 2011 (link); Poland et al. 2012b (link)). DNA was extracted using commercial extraction kits. Restriction/ligation reactions were performed in 96-well plates using 500 ng of DNA from each individual, digestion with HindIII-HF and MspI (New England Biolabs, Ipswich, MA), and 0.1 μM and 10 μM of A1 and A2 adapters per well, respectively. Libraries were pooled, size-selected on a 1% agarose gel, column-cleaned using a PCR purification kit (Qiagen, Valencia, CA), and amplified for 12 cycles using Phusion DNA polymerase (NEB). Average fragment size was estimated on a Bioanalyzer 2100 (Agilent, Santa Clara, CA) using a DNA1000 chip following a second column-cleaning, and library quantification was performed using PicoGreen (Invitrogen, Carlsbad, CA). Pooled libraries were adjusted to 10 nmol and sequenced with 100-bp, single-end reads on the HiSeq2000 (Illumina, San Diego, CA).
Phusion dna polymerase
Manufactured by Qiagen
Phusion DNA polymerase is a high-fidelity DNA polymerase used for PCR amplification. It has a proofreading activity that ensures accurate DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Pcr purification kit, by Qiagen (2 mentions)
Mrna purification kit, by Thermo Fisher Scientific (2 mentions)
Pcr cleanup kit, by Qiagen (2 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Picogreen, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Pierce rna 3 end biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Megascript kit, by Thermo Fisher Scientific (1 mentions)
Pcr cleanup kit, by Illumina (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
5 protocols using phusion dna polymerase
1
GBS Library Construction and Sequencing
2
Biotinylated RNA Probe Synthesis
3
PAS-seq Protocol for Transcriptome Profiling
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PAS-seq was performed as described previously (Zhang et al., 2015) . Briefly, poly(A) + RNAs from 12-day-seedlings were purified using an mRNA purification kit (Invitrogen) and fragmented by heating at 95 C for 25 min. Reverse transcription (Superscript II, Invitrogen) was carried out using our modified HITS-3 0 adaptor at 42 C for 30 min, then the HITS-5 0 adaptor (an SMART oligo) was added and the sample incubated for an additional 30 min. The cDNAs were purified using a Qiagen PCR Cleanup kit and the second-strand cDNAs were synthesized by three cycles of PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. PCR products were separated in a 2.5% agarose gel and 200to 300-base pair (bp) bands were excised and purified. Gel-extracted DNAs were amplified for an additional 13 cycles. PCR products were purified using a Qiagen PCR Cleanup kit and sequenced by Illumina. Oligos for PAS-seq are listed in the Supplemental information, Supplemental Table 6.
4
Parallel Analysis of RNA Sequence (PAS-Seq)
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PAS-Seq was performed as described previously35 (link),36 (link) with modifications at the HITS-3′ adaptor and sequencing primer. Briefly, poly(A) RNAs were purified using an mRNA purification kit (Invitrogen), and fragmented by heating at 95 °C for 30 min. Reverse transcription (Superscript, Invitrogen) was carried out using our modified HITS-3′ adaptor at 42 °C for 30 min, then the HITS-5′ adaptor (a SMART oligo) was added and incubated for an additional 30 min. The cDNAs were purified using a Qiagen PCR Cleanup kit and the second strand cDNAs were synthesized by three cycles of PCR using Phusion DNA polymerase (NEB) and the PE1.0 and PE2.0 primers. PCR products were separated on a 2% agarose gel and 200-300 bp bands were excised and purified. Gel-extracted DNAs were amplified for additional 13 cycles. PCR products were purified using a Qiagen PCR Cleanup kit. TA-cloning was performed before Illumina sequencing. Oligos for PAS-Seq are listed in Supplementary information, Table S4 .
5
Genotyping Mice Embryos and Pups
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Embryos (skull tissues) and pups (tail snips) were genotyped for the HTT transgene and sexed using SRY (sex-determining region Y protein). DNA was extracted (Qiagen kit: # 60506) and PCR amplification was carried out using Phusion® DNA polymerase in combination with the HTT primer pair: (forward) 5′-CCG CTC AGG TTC TGC TTT TA-3′ and (reverse) 5′-TGG AAG GAC TTG AGG GAC TC-3′; or the SRY primer pair: (forward) 5′-TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA-3′, and (reverse) 5′-CCA CTC TGT GAC ACT TTA GCC CTC CGA-3′. Primers were purchased from Invitrogen Life Technologies (New York, NY).
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