Single-cell suspensions were loaded onto 10x Genomics Single Cell 3′ Chips along with the reverse transcription (RT) mastermix per the manufacturer's protocol for the Chromium Single Cell 3′ Library (10x Genomics; PN-120233) to generate single-cell gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: for 2 h at 55°C; for 5 min at 85°C; hold 4°C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; catalog no. 37002D) and SPRIselect beads (Beckman Coulter; catalog no. B23318). Purified cDNA was amplified as follows: for 3 min at 98°C; 12× (for 15 sec at 98°C; for 20 sec at 67°C; for 60 sec at 72°C); for 60 sec at 72°C; hold 4°C. Amplified cDNA was purified using SPRIselect beads and sheared to ∼200 bp with a Covaris S2 instrument (Covaris) using the manufacturer's recommended parameters. Sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples 1–3 and 4–5 were multiplexed, respectively, and sequenced on an Illumina NextSeq 500 (NextSeq control software v2.0.2/Real Time Analysis v2.4.11) using a 150-cycle NextSeq 500/550 High Output Reagent Kit v2 (Illumina, FC-404-2002) in standalone mode as follows: 98 bp (Read 1), 14 bp (I7 Index), 8 bp (I5 Index), and 10 bp (Read 2).
Deep well reaction module
Manufactured by Bio-Rad
The Deep Well Reaction Module is a laboratory equipment designed to provide a controlled environment for various chemical and biological reactions. It features multiple deep wells that can accommodate a range of sample volumes, enabling efficient parallel processing and experimentation. The module is constructed with durable materials to ensure reliable performance in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
C1000 touch thermal cycler, by Bio-Rad (3 mentions)
Nextseq 500 550 high output reagent kit v2, by Illumina (2 mentions)
Chromium single cell 3 library, by 10x Genomics (2 mentions)
Spriselect beads, by Beckman Coulter (2 mentions)
Dynabeads myone silane beads, by Thermo Fisher Scientific (2 mentions)
S2 instrument, by Covaris (2 mentions)
Spriselect beads, by Covaris (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Single cell 3 chip, by 10x Genomics (1 mentions)
Hiseq x pe150, by Illumina (1 mentions)
3 protocols using deep well reaction module
1
Single-Cell Transcriptome Profiling Protocol
2
Single-Cell RNA Sequencing Using 10X Genomics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The scRNA-Seq libraries were prepared from individual cells using the 10X Genomics platform. The Chromium Single Cell 3’ Library & Gel Bead Kit v2 (PN-120237), Chromium Single Cell 3’ Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer’s instructions. Briefly, for each sample, approximately 9000 cells were loaded onto the 10X Genomics Chromium Controller machine for Gel Beads-in-Emulsion (GEM) generation. Reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) using the following program: 55 °C for 2 h; 85 °C for 5 min; hold 4 °C. cDNA was recovered, purified, and amplified to generate sufficient quantities for library preparation. All single-cell libraries were sequenced on the Illumina Hiseq X (PE150).
3
Single-Cell RNA Sequencing Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were loaded onto 10X Genomics Single Cell 3′ Chips along with the RT mastermix as per the manufacturer’s protocol for the Chromium Single Cell 3′ Library (v2, PN-120233; 10X Genomics), to generate single-cell gel beads in emulsion. RT was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: 55°C for 2 h; 85°C for 5 min; hold 4°C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (catalog no. 37002D; Thermo Fisher Scientific) and SPRIselect beads (catalog no. B23318; Beckman Coulter). Purified cDNA was amplified as follows: 98°C for 3 min; 12 times (98°C for 15 s, 67°C for 20 s, 72°C for 60 s); 72°C for 60 s; hold 4°C. Amplified cDNA was purified using SPRIselect beads and sheared to ∼200 bp with a Covaris S2 instrument using the manufacturer’s recommended parameters. Sequencing libraries were generated with unique sample indices for each sample. Libraries for samples 1–3 and 4–5 were multiplexed respectively and sequenced on an Illumina NextSeq 500 (NextSeq control software v2.0.2/ Real Time Analysis v2.4.11) using a 150-cycle NextSeq 500/550 High Output Reagent Kit v2 (FC-404-2002; Illumina) in stand-alone mode as follows: 98 bp (read 1), 14 bp (I7 index), 8 bp (I5 index), and 485 10 bp (read 2).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!