Complementary DNA (cDNA) was prepared using a Sequence Independent Single Primer Amplification (SISPA) approach adapted from Greninger et al. [28 (link)]. Reverse transcription and second strand cDNA synthesis were as described [28 (link)]. cDNA amplification was performed using AccuTaq LA (Sigma, Poole, United Kingdom), in which 5μL of cDNA and 1 μL (100 pmol/μL) Primer B (5′-GTTTCCCACTGGAGGATA-3′) were added to a 50 μL reaction, according to manufacturer's instructions. PCR conditions were 98 °C for 30s, followed by 30 cycles of 94 °C for 15 s, 50 °C for 20 s, and 68 °C for 5 min, and a final step of 68 °C for 10 min. Amplified cDNA was purified using a 1:1 ratio of AMPure XP beads (Beckman Coulter, Brea, California (CA)) and quantified using the Qubit High Sensitivity dsDNA kit (Thermo Fisher, Waltham, US).
Accutaq la
Manufactured by Merck Group
Sourced in United Kingdom
AccuTaq LA is a high-fidelity DNA polymerase designed for use in PCR amplification of long and challenging DNA templates. It exhibits robust performance and increased processivity compared to standard Taq DNA polymerases.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (3 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (2 mentions)
Qubit and high sensitivity dsdna kit, by Thermo Fisher Scientific (1 mentions)
Nebnext rrna depletion kit human mouse rat, by New England Biolabs (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
3 protocols using accutaq la
1
Sequence-Independent Single Primer Amplification for cDNA
2
Single-Cell RNA Sequencing Library Preparation
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The isolated RNA was depleted of ribosomal RNA using NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (New England BioLabs Inc.) and was cleaned up using a Zymo Clean and Concentrator column (Zymo Research). A 4 μl aliquot of RNA was used to prepare complementary DNA (cDNA) using a Sequence Independent Single Primer Amplification approach adapted from ref45 (link). Reverse transcription and second-strand cDNA synthesis were as described. The cDNA amplification was performed using AccuTaq LA (Sigma), in which 5 μl of cDNA and 1 μl (100 pmol/μl) primer B (5′-GTTTCCCACTGGAGGATA-3′) were added to a 50 μl reaction, according to the manufacturer’s instructions. The PCR conditions were as follows: 98 °C for 30 s, 30 cycles of 94 °C for 15 s, 50 °C for 20 s, and 68 °C for 5 min, followed by 68 °C for 10 min. Amplified cDNA was purified using a 1:2 ratio of AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using a Qubit and High Sensitivity dsDNA Kit (Thermo Fisher Scientific Inc.).
3
SISPA-based cDNA Preparation for Viral RNA
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Viral RNA was extracted as described above then Complementary DNA (cDNA) was prepared using a SISPA approach [44 (link)]. In brief, firstly RNA was reverse-transcribed with SuperScript III Reverse Transcriptase (Life Technologies, UK) using Sol-Primer A (5’-GTTTCCCACTGGAGGATA-N9–3’) [45 ]. Then 5μL of cDNA and 1μL (100pmol/μL) Primer B (5’-GTTTCCCACTGGAGGATA-3’) were added to a 50μL reaction using AccuTaq LA (Sigma, Poole, United Kingdom), according to manufacturer’s instructions. PCR conditions were 98°C for 30s, followed by 30 cycles of 94°C for 15s, 50°C for 20 s, and 68°C for 5 min, and a final step of 68°C for 10 min. Amplified cDNA was purified using a 1:1 ratio of AMPure XP beads (Beckman Coulter, Brea, California, US) and quantified using the Qubit High Sensitivity dsDNA kit (Thermo Fisher Scientific, UK).
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