For one responder (patient #003) and one non-responder (patient #008), single-cell RNA-seq and single-cell TCR-seq libraries were prepared using the Single Cell Immune Profiling Solution Kit (10× Genomics, Pleasanton, CA, USA), according to the manufacturer’s instructions. For the gene-expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected with SPRIselect beads (Beckman Coulter), PCR-amplified with sample-indexing primers (10× Genomics, Pleasanton, CA, USA) and double-sided size-selected with SPRIselect beads (Beckman Coulter, Brea, CA, USA). For TCR library construction, TCR transcripts were enriched from 2 μL of amplified cDNA by PCR. Following TCR enrichment, the PCR product was fragmented and end-repaired, size-selected with SPRIselect beads (Beckman Coulter, Brea, CA, USA), PCR-amplified with sample-indexing primers and size-selected with SPRIselect beads (Beckman Coulter, Brea, CA, USA). The single-cell RNA libraries were sequenced on an Illumina HiSeq 4000™ (San Diego, CA, USA). The single-cell TCR libraries were sequenced on an Illumina NextSeq 550™ paired-end 150 mid-output flow cell to a minimum sequencing depth of 5000 reads per cell.
Spriselect beads
Manufactured by Beckman Coulter
Sourced in United States
SPRIselect beads are paramagnetic beads used for nucleic acid purification and size selection. They provide a simple and effective method for isolating DNA or RNA from various sample types. The beads can be used to perform size-selective capture and purification of nucleic acids, enabling efficient removal of contaminants and unwanted fragments.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (27 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (14 mentions)
Hiseq 2500, by Illumina (11 mentions)
Hiseq 4000, by Illumina (11 mentions)
Tapestation 4200, by Agilent Technologies (10 mentions)
End repair mix, by Qiagen (8 mentions)
T4 dna ligase, by Qiagen (8 mentions)
C1000 touch thermal cycler, by Bio-Rad (8 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (8 mentions)
Bioanalyzer, by Agilent Technologies (7 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Dynabeads myone silane beads, by Thermo Fisher Scientific (7 mentions)
Chromium controller, by 10x Genomics (6 mentions)
Klenow exo, by Qiagen (6 mentions)
Miseq platform, by Illumina (6 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Truseq chip sample preparation kit, by Illumina (5 mentions)
Minelute pcr purification kit, by Qiagen (5 mentions)
Quantus fluorometer, by Promega (5 mentions)
188 protocols using spriselect beads
1
Single-Cell Immune Profiling of Responders and Non-Responders
2
Whole Transcriptome RNA-Seq Library Preparation
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High‐throughput whole transcriptome RNA‐sequencing (RNA‐seq) was performed by CD Genomics (Shirley, NY, USA). First, the total RNA was depleted of ribosomal RNA (rRNA) using the Ribo‐Zero HMR kit (Illumina, San Diego, CA, USA) according to the manufacturers instructions. The rRNA‐depleted RNA was then purified using 2x RNAClean XP beads (Beckman Coulter, Brea, CA, USA) followed by first and second‐strand synthesis using NEBNext, New England Biolabs reagents. The generated cDNA was purified with 1.8x SPRIselect Beads (Beckman Coulter). Endprep reaction was performed following the manufacturer's protocol for NEBNext Ultra II End Prep Reaction for the rest of the procedure. Adaptor ligation was then performed using NEBNext Ultra II Ligation protocols (New England Biolabs, Ipswich, MA, USA), and the ligated product was purified by SPRIselect Beads (Beckman Coulter) followed by elution in nuclease‐free water. PCR was carried out using NEBNext Ultra II Q5 Master Mix, and primers and the final library was subsequently purified with SPRIselect Beads. Libraries were sequenced using the Illumina HiSeq platform, generating 2 × 150 bp paired‐end reads and at least 50 million reads per library according to the manufacturer's instructions.
3
Single-Cell Immune Profiling Across Multiple Modalities
4
ATAC-seq protocol for proerythroblasts
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ATAC-seq was performed as previously described40 . In brief, 5 × 104 sorted proerythroblasts were lysed by gently pipetting in cold lysis buffer. Cell lysate was resuspended in a transposition reaction mix (Illumina) and incubated at 37 °C for 30 min. Reactions were purified using AmpureXP beads (Beckman Coulter) following the manufacturer’s protocol with minor changes. Beads were used at a 1:1.1 ratio and reactions were washed twice. After purification samples were amplified using 1 × NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers 1 and 2 (ref. 41 (link)). Libraries were amplified again for an additional 17–19 cycles and left side size selected with SPRIselect beads (Beckman Coulter) at a 1:1 ratio following manufacturer’s protocol, then right side size selected with SPRIselect beads (Beckman Coulter) at a 1:0.5 ratio following manufacturer’s protocol. Library quality was evaluated on a Bioanalyzer and sequencing was performed on a Hiseq2500v2 platform in rapid mode to generate 50 million reads per sample.
5
10x Chromium Single-Cell Library Preparation
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10xChromium single cell libraries were prepared according to the standard protocol outlined in the manual. Briefly, sorted single cell suspension, 10x barcoded gel beads, and oil were loaded into Chromium™ Single Cell A Chip to capture single cells in nanoliter-scale oil droplets by Chromium™ Controller and to generate Gel Bead-In-Emulsions (GEMs). Full length cDNA libraries were prepared by incubation of GEMs in a thermocycler machine. GEMs containing cDNAs were broken and all single cell cDNA libraries were pooled together, cleaned using DynaBeads MyOne™ Silane beads (Fisher PN 37002D), and pre-amplified by PCR to generate sufficient mass for sequencing library construction. Sequencing libraries were constructed by following the steps: cDNA fragmentation, end repair & A-tailing, size selection by SPRIselect beads (Beckman Coulter, PN B23318), adaptor ligation, sample index PCR amplification, and a repeat of SPRIselect beads size selection. The final constructed single cell libraries were sequenced by Illumina Nextseq machine with total reads per cell targeted for a minimum of 50,000.
6
Sequencing Analysis of Msn Mutation
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gDNA was extracted from female uninduced iMLL-ENL and WT C57BL/6 ckit+ cells. Six hundred nanograms of gDNA from ~100,000 cells (200,000 alleles) was used to generate the sequencing libraries for each sample. For the synthesized WT and MUT Msn oligo controls, an input estimated to contain 200,000 copies was used. Amplicons of ∼180 bp containing the region of interest were amplified using PCR with custom primers. The amplified PCR products were cleaned up using SPRIselect beads (Beckman Coulter). A second PCR was then performed to add index and sequencing adapters. The amplified final libraries were cleaned up using SPRIselect beads (Beckman Coulter). Purified libraries were run on Agilent High Sensitivity DNA Kit chip (Agilent Technologies) to verify desired size distribution, quantified by Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and pooled at equimolar concentrations. Pooled libraries were loaded on an Illumina MiSeq Reagent Nano Kit v2 flow cell following protocols for single-end 50-cycle sequencing. Sequencing data were downloaded as FASTQ files that were further assessed with the quality control tool FastQC (v.0.11.2) (62 ), including read count, base quality across reads, and guanine and cytosine (GC) content per sequence. Reads were evaluated at the c.833 position for base calls supporting the c.833C>T mutation.
7
ATAC-seq Library Preparation with Phusion DNA Polymerase
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For ATAC-seq libraries, adapters described in Buenrostro et al. (2013 (link), 2015 ) were used using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific, Cat#F530S) following manufacturer’s instruction with 14 cycles. After purification using 1.5x SPRIselect beads (Beckman-Coulter, Cat#B23319), libraries were sequenced with 2× 100 base pairs on a HiSeq4000 machine (Illumina) following Illumina’s instructions.
8
ATAC-seq Library Preparation with Phusion DNA Polymerase
9
Amplification and Analysis of Cancer Variants
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Ampli1 WGA products were cleaned up with 1.8X SPRIselect Beads (Beckman Coulter) and then quantified using Qubit fluorometer (Life Technologies) according to the manufacturer’s instructions. To analyse cancer gene sequence variants, the Ampli1 Cancer Hotspot Panel Custom Beta adapted from Ion Ampliseq CHP v2 by Menarini Silicon Biosystems covering 2265 COSMIC hotspot regions across 315 amplicons of 48 cancer-related genes commonly mutated in cancer was used as previously described [26 (link)].
10
Spider Probe Kit: Anchored Hybrid Enrichment
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Anchored Hybrid Enrichment data were collected through the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com ) following the general methods of Lemmon et al. [30 (link)] and Prum et al. [59 ]. Briefly, each genomic DNA sample was sonicated to a fragment size of ~175-325 bp using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Subsequently, library preparation and indexing were performed on a Beckman-Coulter Biomek FXp liquid-handling robot following a protocol modified from Meyer and Kircher [60 (link)]. One important modification is a size-selection step after blunt-end repair using SPRIselect beads (Beckman-Coulter Inc.; 0.9x ratio of bead to sample volume). Indexed samples were then pooled at equal quantities (approximately 16 samples per pool), and enrichments were performed on each multi-sample pool using an Agilent Custom SureSelect kit (Agilent Technologies), described herein as the Spider Probe Kit, that contained the probes designed for AHE loci from the spider genomic data detailed above. After enrichment, each set of reactions were pooled in equal quantities for sequencing on three PE150 Illumina HiSeq2500 lanes. Sequencing was performed in the Translational Science Laboratory in the College of Medicine at Florida State University.
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