Six small RNA libraries (Q1, Q2, T1, T2, B1 and B2) were constructed and sequenced with Illumina TruSeq deep sequencing technology (Sample Preparation Guide, Par #15004197 Rev.A, Illumina, San Diego, CA). Briefly, small RNAs (18–30 nt) were size-selected by gel fraction and extracted by centrifugation. After ligation of 5′ and 3′ adaptors, small RNAs were reverse transcribed into cDNA, then amplified using the sequencing primers for 14 cycles and the fragments (~ 150 bps) were isolated from a 6% TBE PAGE-gel. After the cDNA was purified, it was used for cluster generation and sequenced using an Illumina HiSeq 2000 platform. Image files generated by the sequencer were processed to nucleotide sequences (raw FASTQ files) using a base-calling pipeline (Illumina). FASTQ files for all six libraries have been submitted to Sequence Read Archive (SRA) of NCBI under the accession number SRA347706 (Q1: SRR3184695, Q2: SRR3184696, T2: SRR3184697, T1: SRR3184698, B1: SRR3184699, and B2: SRR3184700).
Truseq deep sequencing technology
Manufactured by Illumina
The TruSeq deep sequencing technology is a high-throughput DNA sequencing platform developed by Illumina. It uses a proprietary sequencing-by-synthesis approach to generate large volumes of high-quality, accurate sequence data.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000 platform, by Illumina (2 mentions)
Truseq small rna sample prep kit, by Illumina (2 mentions)
Agilent 2200 tapestation, by Agilent Technologies (2 mentions)
Base calling pipeline, by Illumina (1 mentions)
Tbe polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 100, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using truseq deep sequencing technology
1
Illumina TruSeq small RNA sequencing
2
Small RNA Sequencing of Extracellular Vesicles
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Four small RNA libraries (from parental LIM1863 cells and derived sMVs, A33-, and EpCAM-Exos) were constructed and sequenced with Illumina TruSeq deep sequencing technology (Sample Preparation Guide, Par #15004197 Rev.A, Illumina, San Diego, CA). Briefly, total RNA samples were fractionated on a 15% Tris-borate-EDTA (TBE) polyacrylamide gel (Invitrogen) and a bands corresponding to small RNAs (18∼30 nt) were excised and small RNAs extracted by centrifugation. After ligation of 5′(5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′) and 3′(5′-UGGAAUUCUCGGGUGCCAAGG-3′) adaptors, small RNA molecules were reverse transcribed into cDNA, then amplified using the adaptor primers for 14 cycles and the fragments (∼150 bps) were isolated from a 6% TBE PAGE-gel. The purified cDNA was directly used for cluster generation and sequenced using an Illumina HiSeq 2000 platform. Image files generated by the sequencer were processed to produce digital-quality data (raw FASTQ files). FASTQ files for all four small RNA libraries (LIM1863 cells, and derived sMVs, A33-Exos and EpCAM-Exos) have been submitted to Sequence Read Archive (SRA) of NCBI under the accession number SRA106214.
3
Profiling Small RNA Expression in CSCs and DCs
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The purified exosomes from CSCs and DCs were separately extracted for total RNA including the small RNA fraction using standard RNA extraction methodology. The quality and quantity of the isolated RNA was determined by the ND100 Nanodrop (Thermo Fisher), while RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA) using an RINe above 7.0. Two small RNA libraries were constructed and sequenced with Illumina TruSeq deep sequencing technology. RNAs were ligated with 3’ RNA adapter, followed by a 5’ adapter ligation. Subsequently, the adapter-ligated RNAs were subjected to RT-PCR and amplified with a low cycle. Then the PCR products were size-selected by PAGE gel according to instructions of TruSeq® Small RNA Sample Prep Kit (Illumina, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and diluted to 10 pM for cluster generation in situ on the HiSeq2500 single-end flow cell, followed by sequencing (1#x00D7;50 bp) on HiSeq 2500 platform. Image files generated by the sequencer were processed to produce digital quality data (raw FASTQ files).
4
Small RNA Sequencing of SOX2 Overexpression
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The DMS114 cells both before and after SOX2 overexpression were separately extracted for total RNA, including the small RNA, fraction using Trizol reagent (Invitrogen). The quality and quantity of the isolated RNA were determined by using an ND‐1000 Nanodrop UV‐Vis spectrophotometer (Thermo Fisher Scientific), while RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies) using an RNA integrity number equivalent >7.0. Two small RNA libraries were constructed and sequenced using Illumina TruSeq deep sequencing technology and a TruSeq® Small RNA Sample Prep Kit (Illumina) according to the manufacturer's instructions. Briefly the RNAs were ligated with a 3ʹ RNA adapter, followed by a 5ʹ adapter ligation. Subsequently, the adapter‐ligated RNAs were subjected to reverse transcription‐PCR and amplified with a low number of cycles. Then, the PCR products were size‐selected by polyacrylamide gel electrophoresis (PAGE). The purified library products were evaluated using the Agilent 2200 TapeStation and diluted to 10 p.m. for cluster generation in situ on the HiSeq 2500 single‐end flow cell, followed by sequencing (1#x00D7;50 bp) on a HiSeq 2500 platform. The image files generated by the sequencer were processed to produce digital quality data (raw FASTQ files).
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