title: GSM4503585: TO1000 Rep 1 RNA-seq; Brassica oleracea; RNA-Seq
library_construction_protocol: Lysis and homogenization of fresh soft tissue were performed before RNA extraction; PureLink Micro-to-Midi Total RNA Purification System (Invitrogen, Cat. No 12183-018, Carlsbad, California, USA) was used. A total of eight plants were pooled per replicate in a single tube for a total of eight plants per replicate. RNA extraction was done using the Purelink RNA Mini Kit (Ambion, Cat. No12183-018A, Carlsbad, California, USA). DNase was not used before proceeding with cDNA synthesis as recommended in the protocol. For cDNA synthesis MINT (Evrogen, Cat. No SK001, Moscow, Russia) was used, this kit is used to synthesize full-length-enriched double stranded cDNA from a total polyA+ RNA. The cDNA was then amplified by 16 -- 21 cycles of polymerase chain reaction and purified using a PCR Purification Kit (Invitrogen K3100-01 Carlsbad, California, USA). Protocols previously published by Steele et al. (2012) were used. End repair was performed on 400ul with 3ug of normalized ds-cDNA prior to ligating barcoding adapters for multiplexing, using NEB Prep kit E600L (New England Biolabs, Ipswich, Massachusetts, USA). Shearing was done using a Bioruptor® sonication device (Diagenode, Denville New Jersey), at 4°C, continuously, for 15min. total (7mix) on high. The three replicates per morphotype were tagged with different adaptors and sequenced on a single lane. Oligonucleotides used as adapters were: Cabbage (1: ACGT, 2: GCTT, 3: TGCT), Kale (1: TACT, 2:ATGT, 3:GTAT), TO1000 (1:CTGT, 2:AGCT, 3:TCAT). To prepare the adapters, equal volumes were combined of each adapter at 100 μmol/L floating them in a beaker of boiling water for 30min. and snap cooling them on ice. Ligation products were run on a 2% low-melt agarose gel and size-selected for ~300 bp using an x-tracta Disposable Gel Extraction Tool (USA Scientific, Ocala Florida). Fragments were enriched using PCR in 50 μL volumes containing 3 μL of ligation product, 20 μL of ddH2O, 25 μL master mix (from NEB kit), and 1 μL each of a 25 μmol/L solution of each forward and reverse primer (forward 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC* T-3′; reverse 5′-CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC*-3′; both HPLC purified). Thermal cycle routine was as follows: 98°C for 30 s, followed by 15 cycles of 98°C for 10 s, 65°C for 30 s, and 72°C for 30 s, with a final extension step of 72°C for 5 min. Products were run on a 2% low-melt agarose gel and the target product was excised and purified with the tools described above. DNA was purified at each step during the end repair, adapter ligation, size selection and fragment enrichment with either a Gel Extraction kit (Qiagen, Germantown Maryland) or a DNA QIAquick PCR Purification kit (Qiagen, Germantown Maryland).
attributes.tag: GEO Accession
attributes.value: GSM4503585