We designed our SELEX library to contain a 23-bp random region flanked by primer binding regions conforming to the Illumina TruSeq small RNA format for a total of 70 bp. The library was ordered in 1 mmol format from IDT as single strand with handmix option over the randomized region. The complementary strand was synthesized by Klenow extention using 5′-Cy5 labeled TSSR1 primer (Supplemental Table S1 ) on a PCR machine. Briefly, a reaction containing 2.5 µM ssDNA library, 5 µM 5′-Cy5-TSSR1, 150 µM dNTPs in NEB buffer 2 was incubated at 94°C for 3 min and then gradually cooled down to 37°C over 45 min. Six units of Klenow were added to every 25 µL of reaction, incubated at 37°C for 60 min, 72°C for 20 min, and gradually cooled down to 10°C over 45 min. dsDNA was purified using a MinElute PCR column (Qiagen) and quantified by absorbance at 260 nm.
Minelute pcr column
Manufactured by Qiagen
Sourced in Germany
The MinElute® PCR Purification Kit is a spin-column-based system for rapid purification of PCR products. The kit utilizes a silica-membrane technology to efficiently bind DNA fragments from 70 bp to 4 kb, allowing for efficient removal of primers, nucleotides, enzymes, and other impurities.
Automatically generated - may contain errors
Lab products found in correlation
Dynamag magnet, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
Dneasy blood and tissue kit protocol, by Qiagen (1 mentions)
Polynucleotide kinase, by New England Biolabs (1 mentions)
T4 dna ligase reaction buffer, by New England Biolabs (1 mentions)
Klenow fragment, by New England Biolabs (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
5 protocols using minelute pcr column
1
Illumina TruSeq SELEX Library Synthesis
2
Bacterial 16S rRNA Sequencing from BAL
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Raw BALs supernatants were spun at 10,000g and frozen at −80 °C. Pellets were resuspended in 180 μl of freshly prepared lysozyme solution (20 mg/ml lysozyme, 20 mM Tris-Cl, pH 8.0, 2 mM Na-EDTA, 1.2 % Triton X-100, filtered through a 0.22-μm filter) and incubated at 37 °C for 10 min. Extraction followed the Qiagen DNEasy Blood and Tissue kit protocol. Reagent controls, without BAL, were also processed through the Qiagen procedure. Extracted DNA was used in a PCR reaction with Roche/454 GS-FLX sequencing adapter fusion primers targeting the V1–V3 region of the bacterial 16S ribosomal RNA (rRNA). The reaction mix consisted of 0.75 U Accuprime Taq Hifi, 1× Accuprime PCR buffer II, 1 μl DNA sample, and 200 nmol of barcoded primer. Cycling conditions were 95 °C for 2 min, then 30 cycles of 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 5 min. Reactions were cleaned up using 36 μl of Ampure Beads (Agencourt) and eluted into 25 μl low TE, pH 8.0. Eluted PCR products were quantitated in triplicate using DNA Picogreen (Invitrogen). Two final sample pools were created by combining 103 and 104 total PCRs; each positive sample had 20 ng added, while each negative control sample (buffer) had 20 μl added. The combined pools were then purified on a MinElute PCR column (Qiagen) and eluted into TE buffer. A total of 170 BAL samples were multiplexed in this manner in two separate gasket wells.
3
Open Chromatin Profiling via ATAC-seq
4
ChIP-seq Library Preparation Protocol
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ChIP DNA was end repaired by incubation at 20°C for 30 minutes with 3 U of T4 DNA Polymerase (NEB, Hitchin, UK), 10 U of Polynucleotide Kinase (NEB), 2 U DNA Polymerase I Large (Klenow) fragment (NEB), 1× T4 DNA ligase reaction buffer (NEB) and 400 nM dNTPs. The enzymes were then heat inactivated at 75°C for 20 minutes, after which the DNA was ethanol precipitated. A tail of 'A' bases was added to the 3’ ends of the DNA by incubation with 5 U Klenow Fragment (3’-5’ exo-; NEB), 200 nM dATP and 1X buffer 2 (NEB) at 37°C for 30 minutes. The enzymes were heat inactivated and cleaned up as before. Barcoded Ilumina paired end adaptors were then ligated to the processed ChIP DNA by incubation with 300 U of T4 DNA ligase (NEB), 1× T4 DNA ligase buffer (NEB), 7.5% PEG-6000 and 2 pmol of annealed Illumina adaptors for 3 h at room temperature. Ligated DNA was purified using MinElute PCR columns (Qiagen, Hilden, Germany) and eluted in 10 μl water.
5
Open Chromatin Profiling via ATAC-seq
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In order to profile open chromatin regions, stimulated cells were washed with RPMI and Dynabeads® were removed using a DynaMag® magnet (Thermo Fisher). Next, tagmentation was performed using the fast ATAC protocol 39 (link). Briefly, 50,000 cells were washed in cold PBS buffer and resuspended in 50 μl of Nextera® tagmentation buffer supplemented with 0.01% digitonin and 2.5 μl Tn5 transposase (Nextera). Samples were then incubated at 37°C and 800 rpm for 30 minutes. After tagmentation, DNA was purified using MinElute® PCR columns (Qiagen) according to the manufacturer’s instructions and stored at -80°C until library preparation.
Sequencing libraries were generated from tagmented DNA using the Nextera® DNA library preparation kit according to the manufacturer’s instructions. Briefly, DNA was amplified by PCR and fragments of inappropriate sizes were removed using Agencourt AMPure XP beads (BD). Finally, samples were pooled and loaded into an Illumina® HiSeq 2500 for paired-end sequencing. In order to minimise batch effects, samples were randomized before library preparation and before sequencing. We obtained on average 58 million paired-end reads per sample.
Sequencing libraries were generated from tagmented DNA using the Nextera® DNA library preparation kit according to the manufacturer’s instructions. Briefly, DNA was amplified by PCR and fragments of inappropriate sizes were removed using Agencourt AMPure XP beads (BD). Finally, samples were pooled and loaded into an Illumina® HiSeq 2500 for paired-end sequencing. In order to minimise batch effects, samples were randomized before library preparation and before sequencing. We obtained on average 58 million paired-end reads per sample.
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