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22 protocols using oligotex kit

1

Tissue Transcriptomics across Drosophila Development

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Tissues were dissected from Oregon R larval, pupal and adult staged animals synchronized with appropriate age indicators. Pupal and adult animals were treated with a number of environmental stresses. RNA was isolated using TRIzol (Invitrogen), DNased, and purified on a RNAeasy column (Qiagen). poly(A)+ RNA was prepared from an aliquot of each total RNA sample using an Oligotex kit (Qiagen).
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2

Comprehensive Molecular Analysis Protocol

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Propofol (Plofed 1%, Polfa, Poland), quinaprilat (Pfizer, Germany), enalaprilat (Sigma, Poland), lipofundin (MCT/LCT 10%, Braun, Germany), aqua pro injection (Baxter, Poland), sodium hydroxide (Sigma, Poland), ethylenediaminetetraacetic acid (EDTA) (Sigma, Poland), low-serum growth supplement (LSGS, Cascade Biologics, UK), Medium 200 (M200, Cascade Biologics, UK), penicillin (Sigma, Poland), streptomycin (Sigma, Poland), trypsin (Sigma, Poland), trypan blue (Sigma, Poland), Oligotex Kit (Qiagen, USA), qPCRTM Mastermix, SYBR Green I (Eurogentec Seraing, Belgium), TaqMan Reverse Transcription Reagents Kit (Applied Biosystems, USA), Tris Buffer (Polish Chemical Reagents, Poland) and Trizol (Invitrogen Life Technologies, USA) were used in the study.
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3

Evaluation of Hemostasis and Oxidative Stress

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Pro (Plofed 1%, Polfa S.A., Poland), Quin (Accupro, Pfizer, Germany), lipofundin (MCT/LCT 10%, Braun, Germany), gum arabic (Pharma Cosmetic, Poland), pentobarbital (Vetbutal, Biovet, Poland), collagen type I (Collagen, Chronolog, USA), and heparin (Heparinum, Polfa, Poland) were used in the study. Bovine albumin, apyrase, HEPES (N-(2-hydroxyethyl)piperazine-N′-(2-ethanosulfonic acid) were delivered by Sigma-Aldrich (Poland). Calcium chloride, EDTA, glucose, magnesium chloride, sodium chloride, potassium chloride, sodium bicarbonate, sodium phosphate, Tris buffer, and trisodium citrate were provided by Polish Chemicals Reagents (Poland). Rabbit anti-rat TAFI monoclonal antibody (ImmunoKontact AMS Biotechnology, Germany), rat active PAI-1 ELISA kit (Innovative Research, USA), rat active t-PA ELISA kit (Innovative Research, USA), hydrogen peroxide colorimetric detection kit (Assay Designs, USA), correlate assay nitric oxide NO-2/NO-3 assay kit (Assay Designs, USA), MDA adducts ELISA kit (Cell Biolabs, USA), Trizol (Invitrogen Life Technologies, USA), qPCRTM Mastermix, SYBR Green I (Eurogentec Seraing, Belgium), Oligotex Kit (Qiagen, USA), and TaqMan reverse transcription reagents kit (Applied Biosystems, USA) were also used.
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4

Quantifying RSV Gene Expression in Vero Cells

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Vero cells were plated the day before infection in six-well plates. RSV A2 was incubated with 100 µg/mL of either SBA or vehicle control. Cells were then infected with the different virus treatments at an MOI of 1 for 24 h. Total RNA was extracted using an RNeasy kit (Qiagen) following the manufacturer’s protocol. mRNA was then isolated from total RNA using an Oligotex kit (Qiagen) following the manufacturer’s protocol. cDNA synthesis was performed using the same amount of mRNA per condition and an RT2 First Strand kit (Qiagen) following the manufacturer’s protocol. qRT-PCR was then performed in triplicate using the RT2 SYBR Green ROX qPCR Mastermix (Qiagen) and primer sets (Supplementary Table 1) described in Boukhvalvoa et. al.57 (link) on a Step-One Plus real-time thermocycler (Applied Biosystems). Rhesus Macaque GAPDH primers (Qiagen) were used as a loading control. Relative quantitation was performed using the Applied Biosystems software. Infections and extractions were repeated twice.
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5

Quantifying MGMT Transcript Levels by qPCR

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qPCR data were described previously [17 (link)]. Total RNA was isolated using a Qiagen RNeasy kit, and mRNA was subsequently isolated using a Qiagen Oligotex kit according to the manufacturer’s protocols. Following DNase digest, cDNA was generated using poly-dT primers with reverse transcriptase. TaqMan qPCR was used to quantitate MGMT transcript levels relative to a GAPDH control. Primers and probes for MGMT (catalog number Hs.00172470) and GAPDH (Hs.99999905) were purchased from Applied Biosystems. A 20 μL reaction containing TaqMan Universal PCR Master Mix (Applied Biosystems), plus probes and cDNA was amplified by PCR using the following program: 10 minutes at 95°C, followed by 40 cycles of denaturing at 95°C for 15s followed by annealing and extension for 1 minute at 60°C.
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6

T Cell Receptor Repertoire Analysis

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The isolated PBMCs were washed three times with ice-cold PBS then 1 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was added to extract the total RNA according to the manufacturer's instructions. An Oligotex kit (Qiagen, Inc., Valencia, CA, USA) was used to purify the mRNA from total RNA according to the manufacturer's protocol. The purified mRNA was converted to cDNA using SuperScript III Reverse Transcriptase, DNase, buffers and dNTPs (all from Thermal Fisher Scientific, Inc.). A degenerated V region primer (5′-GCIITKTIYTGGTAYMGACA-3′), which was designed to cover the majority of the TRBV genes, and a reverse primer (5′-GCACCTCCTTCCCATTCAC-3′) covering both TRBC genes was used as previously reported (18 (link),19 (link)). Each 50 µl RT-qPCR reaction contained 2 µl cDNA, 12 µl ddH2O, 25 µl Premix Ex Taq (Takara Bio, Inc., Otsu, Japan), 1 µl forward primer, 1 µl reverse primer. The RT-qPCR cycling conditions were used as follows: 94°C for 10 min; 40 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 30 sec; and a final 10 min extension at 72°C and 4°C hold for 30 sec. The PCR reaction was performed using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.).
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7

Quantitative Northern Blot Analysis

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Total RNA was extracted from growing MCF7 cells as described and enriched for mRNA using Oligotex kit (QIAGEN). Equal volumes of reconstituted RNA were separated on 1.2% agarose gel supplemented with 0.66% (vol/vol) formaldehyde and transferred onto Amersham Hybond-N+ membrane (GE Healthcare). RNA probes targeting Renilla or Firefly mRNAs were produced by in vitro transcription (T7 high yield kit, NEB) of PCR fragments encoding for sequences complementary to these transcripts (see Table S4 for used oligos) using 32P(α)-UTP. Following cleaning of the probes using MicroSpin G-50 columns (GE Healthcare), hybridization was performed in ULTRAhyb Hybridization buffer (Ambion) rotating at 68°C for 4 hr. The membrane was washed twice with 2xSSC buffer supplemented with 0.1%SDS and twice with 0.2xSSC buffer supplemented with 0.1%SDS. The signal was detected by FujiFilm FLA-3000 PhosphorImager and analyzed using ImageJ.
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8

Normalized cDNA Libraries and 454 Pyrosequencing

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The construction of two normalized cDNA libraries and 454 pyrosequencing was carried out at the W.M. Keck Center for Comparative and Functional Genomics, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. We chose to normalize the libraries in order to increase the likelihood that rare transcripts would be sequenced, leading to a more complete transcriptome with limited sequencing effort. For each library, messenger RNA was isolated from 10 μg of pooled total RNA with the Oligotex kit (Qiagen, Valencia, CA). The messenger RNA was then converted to a primary cDNA library with adaptors compatible with the 454 system using Multiplex Identifier (MID) tags to distinguish the two population pools
[28 (link)]. The libraries were diluted to 1 × 106 molecules/μL, pooled, and sequenced on a full plate using the 454 Genome Sequencer FLX + system according to the manufacturer’s instructions (454 Life Sciences, Branford, CT). Signal processing and base calling were performed using the bundled 454 Data Analysis Software v2.6.
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9

Peanut Gynophore RNA Extraction and RLM-5' RACE

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Total RNA (200 μg) from peanut gynophore was extracted using CTAB reagent and mRNA was purified using the Oligotex kit (Qiagen). RNA ligase-mediated rapid amplification of 5′ cDNA ends (RLM-5′ RACE) was performed with the RLM-RACE kit according to the manufacturer’s instructions (Clontech). The final PCR product was extracted and purified from 2% agarose gel, cloned into pMD18-T simple vector (Takara). Plasmid DNA from 10 different colonies was sequenced. Gene specific primers used for RLM-5′ RACE experiments were listed in Additional file 8: Table S4.
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10

Degradome Sequencing of Brassica napus

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Degradome libraries were constructed based on a previous method34 . Briefly, poly (A) RNA was extracted from total RNA sample using the Oligotex kit (Qiagen). Polyadenylated transcripts possessing 5′ monophosphates were ligated to RNA adapters consisting of a MmeI recognition site at its 3′ end. After ligation, the first-strand cDNA was generated using oligo d(T) and amplified using five PCR cycles. The PCR product was purified and digested with MmeI. The digested PCR product was then ligated to a 3′ double DNA adapter, amplified by 20 PCR cycles, and gel-purified for Illumina sequencing.
Sequenced tags with 18–21 nucleotides long were normalized after trimming sequence adapters and filtering the low quality tags. The sliced miRNA targets were identified and classified based on the CleaveLand pipeline33 (link). The unique reads were normalized to give reads per million and subsequently mapped to annotated cDNA sequences from the Gene Index database (ftp://occams.dfci.harvard.edu/pub/bio/tgi/data/Brassica_napus) (BnGI release 5.0).
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