Total RNA was isolated from cells using TRIzol (Invitrogen). A KAPA Stranded mRNA-Seq Kit (KAPA) was used following the manufacturer’s instructions. Adapters were offered by a TruSeq Library Prep Pooling kit (Illumina). Paired-end 150-bp sequencing was further performed on a NovaSeq 6000 (Illumina) at Berry Genomics Corporation.
Truseq library prep pooling kit
Manufactured by Illumina
Sourced in United States
The TruSeq Library Prep Pooling kit is a laboratory equipment product designed for use in next-generation sequencing (NGS) workflows. The kit provides a standardized method for pooling libraries prior to sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Hiseq 2000 or 2500, by Illumina (3 mentions)
Kapa stranded mrna seq kit, by Roche (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Bsa pbs, by Merck Group (2 mentions)
Pronase e, by Merck Group (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (1 mentions)
6 protocols using truseq library prep pooling kit
1
Transcriptome Profiling via RNA-Seq
2
Single-Cell Histone Modification Profiling
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For ULI-NChIP-seq, 500–1000 cells were used per immunoprecipitation reaction. SCNT or WT embryos in two-cell stage were treated with 0.5% Pronase E (Sigma) to remove the zona pellucida. And the polar bodies were removed by gently pipetting in a pipette after incubation with Ca2+-free CZB. CCs were collected following the protocol of donor-cell preparation for SCNT. All isolated cells were washed three times in 0.5% BSA-PBS (Sigma) solution to avoid any possible contaminant. The procedure of ULI-NChIP was carried out as previously described [18 (link)]. And 1.5 μg of histone H3K9me3 antibody (Active Motif, Carlsbad, CA, USA, 39161) were used for each immunoprecipitation reaction.
The sequence libraries were generated using the TruSeq Library Prep Pooling kit (Illumina 15042173), following the manufacturer’s instructions. Paired-end 100-bp sequencing was further performed on HiSeq 2500 or 2000 (Illumina) at the National Institute of Biological Sciences and Peking University.
The sequence libraries were generated using the TruSeq Library Prep Pooling kit (Illumina 15042173), following the manufacturer’s instructions. Paired-end 100-bp sequencing was further performed on HiSeq 2500 or 2000 (Illumina) at the National Institute of Biological Sciences and Peking University.
3
RNA-seq of Induced Murine Fibroblasts
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Total RNA from independent biological replicates of each uninduced MEF, induced MEFs with or without Obox1 at 72 hr for OSKM + Obox1 system, and of overexpressing OSKM, OKM + Obox1, or OKM + empty vector at 72 hr for replacement system, was isolated using the QIAGEN RNeasy Kit (Germantown, USA). The RNA samples were subject to mRNA fragmentation, cDNA synthesis, and library preparation using a KAPA Stranded RNA-Seq Kit Illumina platform (KK8440; Kapa, Wilmington, USA). All adapters were diluted from the adapters offered by TruSeq Library Prep Pooling kit (Illumina, USA). Single-end 50-bp sequencing was further performed on HiSeq 2500 (Illumina) at Berry Genomics.
4
Total RNA Isolation and Sequencing
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Total RNA was isolated from cells using TRIzol (Invitrogen). To generate RNA-sequencing libraries, a KAPA stranded mRNA-Seq kit (KAPA) was used following the manufacturer’s instructions. Adapters were through a TruSeq Library Prep Pooling kit (Illumina). Paired-end150 bp sequencing was further performed on a Novaseq 6000 (Illumina) at Berry Genomics Corporation.
5
RNA-seq of iPSC Reprogramming
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Total RNAs were isolated from naïve iPSCs and reprogramming cells using TRizol (Invitrogen). To generate RNA sequencing libraries, KAPA Stranded mRNA-Seq Kit (KAPA) was used following the manufacturer’s instructions. Adapters were offered by TruSeq Library Prep Pooling kit (Illumina). Single-end 50 bp sequencing was further performed on a HiSeq 2500 or 2000 (Illumina) at Berry Genomics Corporation.
6
Single-Cell RNA-Seq Protocol for Early Embryos
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The single-cell RNA-seq method followed previously published studies [13 (link), 30 (link)]. In brief, the harvested single blastomeres, ICM or TE, were washed several times in 0.5% BSA-PBS (Sigma) solution and subsequently picked and transferred into lysate buffer by a mouth pipette. Diluted ERCC mix (Life Technologies 4456740) was added in lysis buffer as spick-in for each sample. Reverse transcription was performed directly on the cytoplasmic lysate. Terminal deoxynucleotidyl transferase was then used to add a poly(A) tail to the 3′ end of the first-strand cDNAs. The total cDNA library of the single cell was then amplified by 18–20 cycles for the library construction.
The amplified cDNA was fragmented using Covaris sonicator (Covaris S220, Woburn, MA, USA). To generate the sequence libraries, the TruSeq Library Prep Pooling kit (Illumina 15042173, San Diego, CA, USA) or NEBNext Ultra DNA Library Prep Kit (New England Biolabs E7370, Ipswich, MA, USA) was used following the manufacturer’s instructions. All adapters with index-barcode in this project also including the RRBS and ULI-NChIP-seq were diluted (or not) from the adapters offered by TruSeq Library Prep Pooling kit. Paired-end 100-bp or 125-bp sequencing was further performed on a HiSeq 2500 or 2000 (Illumina) at the National Institute of Biological Sciences, Peking University and Berry Genomics Corporation.
The amplified cDNA was fragmented using Covaris sonicator (Covaris S220, Woburn, MA, USA). To generate the sequence libraries, the TruSeq Library Prep Pooling kit (Illumina 15042173, San Diego, CA, USA) or NEBNext Ultra DNA Library Prep Kit (New England Biolabs E7370, Ipswich, MA, USA) was used following the manufacturer’s instructions. All adapters with index-barcode in this project also including the RRBS and ULI-NChIP-seq were diluted (or not) from the adapters offered by TruSeq Library Prep Pooling kit. Paired-end 100-bp or 125-bp sequencing was further performed on a HiSeq 2500 or 2000 (Illumina) at the National Institute of Biological Sciences, Peking University and Berry Genomics Corporation.
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