Aug 21, 2025
scQers library preparation
This protocol is a draft, published without a DOI.
- Jean-Benoît alanne1
- 1Department of Genome Sciences, University of Washington, Seattle, WA, USA
Protocol Citation: Jean-Benoît alanne 2025. scQers library preparation. protocols.io https://protocols.io/view/scqers-library-preparation-g74zbzqx7
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Full methods can be found in the Supplementary Information from Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters
Created: August 20, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 225145
Keywords: function multicellular expression phenotypes from cre variant, cell quantitative expression reporter, multicellular expression phenotype, rna barcode stabilization via circularization, specific developmental cre, cell reporter assay, accessible chromatin, perturbed transcription factor, quantitative expression reporter, transcription factor, regions of accessible chromatin, dual rna cassette, measurement of reporter expression, rna, multicellular, bottleneck in genomic, cre variant, genomic, regulatory element, synthetic cres at scale, quantification tasks inherent to multiplex, chimeric cre, cre, synthetic cre
Funders Acknowledgements:
NHGRI
Grant ID: UM1HG011966
NHGRI
Grant ID: R01HG010632
Damon Runyon Cancer Research Foundation
Grant ID: DRG-2435-21
NHGRI
Grant ID: F31HG011576
National Heart, Lung, and Blood Institute
Grant ID: T32HL007828
NHGRI
Grant ID: F32HG011817
Abstract
The inability to scalably and precisely measure the activity of developmental cis-regulatory elements (CREs) in multicellular systems is a bottleneck in genomics. Here we develop a dual RNA cassette that decouples the detection and quantification tasks inherent to multiplex single-cell reporter assays. The resulting measurement of reporter expression is accurate over multiple orders of magnitude, with a precision approaching the limit set by Poisson counting noise. Together with RNA barcode stabilization via circularization, these scalable single-cell quantitative expression reporters provide high-contrast readouts, analogous to classic in situ assays but entirely from sequencing. Screening >200 regions of accessible chromatin in a multicellular in vitro model of early mammalian development, we identify 13 (8 previously uncharacterized) autonomous and cell-type-specific developmental CREs. We further demonstrate that chimeric CRE pairs generate cognate two-cell-type activity profiles and assess gain- and loss-of-function multicellular expression phenotypes from CRE variants with perturbed transcription factor binding sites. Single-cell quantitative expression reporters can be applied in developmental and multicellular systems to quantitatively characterize native, perturbed and synthetic CREs at scale, with high sensitivity and at single-cell resolution.
Troubleshooting
Single-cell reporter libraries preparation from the methods in: Multiplex profiling of developmental cis-regulatory elements with quantitative single-cell expression reporters
For single-cell reporters, three libraries are generated: the standard 3’ gene expression (GEx) library from 10x, and two custom derived libraries, one for each reporter RNA (oBC and mBC). The latter are obtained from nested PCRs from the amplified cDNA as we detail below.
Briefly, single-cell library preparation proceeded following the manufacturer's protocol (v3.1 manual CG000205 Rev D, 10x Genomics), with some critical modifications listed here. First, one of the replicate’s cDNA (replicate B) was split in two equal halves (and brought to same final volume with elution solution 1) after GEM RT cleanup (step 2.1.s) prior to cDNA amplification to allow for a direct comparison the UMIs captured with different enrichment strategy (hereafter replicate B1 and B2). For cDNA amplification, primers specific to the mBC (oSR38) and oBC (oJBL246) reporter transcripts were spiked-in the reaction (similar to TAP-seq (44)) at final concentration of 0.5 uM to boost UMI capture for replicates A and B1 (but not for replicate B2, to allow direct comparison with replicate B1). Following cDNA amplification, both the bead and supernatant derived material (steps 2.3Ax and 2.3Bxiv respectively) were saved for downstream processing.
Gene expression libraries for all replicates were prepared following the manufacturer’s protocol from 25% of the bead fraction amplified cDNA.
oBC enriched libraries were prepared as follows. For replicate B2 (no primer spiked in), a first outer PCR1 was performed using 25% of the supernatant amplified cDNA with primers oSR40+oJBL246 using Kapa Robust (Roche) and tracking with qPCR until the inflection point (50 uL 2x master mix, 12.5 uL supernatant cDNA, 5 μL 10 μM oJBL246, 5 μL 10 μM oSR40, 0.5 uL SYBr green, and water to 100 μL; run parameters: 3 min at 95C, and cycles 20 s at 95C, 20 s at 60C, 20 s at 72C). Amplicons were cleaned up with 1.75x Ampure XP beads, and 1/10 of the eluate was carried to the inner PCR with the remaining replicates. For replicates A and B1, the outer PCR was performed during the cDNA amplification via the spiked-in primer, and 25% of the supernatant amplified cDNA was taken as input for the next PCR. Semi-nested inner PCR was performed on all samples with primers NextP5_index1 and indexed primers oJBL425-oJBL427, with the same parameters as PCR1 and stopped before the inflection point. Final libraries were purified by 1.5x Ampure XP beads.
As a result of our Pol II reporter construct having a capture sequence (CS2, Fig. S1A) downstream of the mBC, reporter mRNAs could be captured from both the poly-dT and CS2 reverse transcription primers on the 10x beads. To systematically compare capture efficiency resulting from the two types of primers, two different libraries were generated (poly-dT captured, and CS2 captured). For poly-dT captured libraries, similar to oBC libraries, we first performed outer PCR on replicate B2 (no spiked-in primers in cDNA amplification) using primers oSR38+oJBL207, using the same PCR conditions as for oBC except for an elongation time of 50 s and an anneal temperature of 65C. 25% of the bead fraction of the purified amplified cDNA was used as template. Following 1x Ampure XP clean up, 10% of the eluate was taken for PCR2. PCR2 was performed on all replicates (directly using 25% of the bead-derived amplified cDNA for replicates A and B1) using primers oJBL324+oJBL495 and the same parameters as PCR1, tracking by qPCR and purifying by 1x Ampure XP beads. A final PCR was performed to index amplicons with primers oJBL076 and indexed primers (oJBL496-oJBL498), and the resulting amplicons purified by 1x Ampure XP beads. The CS2 libraries were prepared entirely analogously to poly-dT captured libraries, except with the following primers: PCR1 for replicate B2 (SR38+SR40), PCR2 all replicates (oJBL529+oSR40), PCR3 all replicates (NextP5_index1+ indexed primers oJBL530-oJBL532).
We note that for both mBC and oBC libraries, semi-nested PCR is necessary to obtain a clean amplicon library (multiple non-specific amplification products were visible following the outer PCR, but a highly specific product was obtained following the semi-nested inner PCR).
All libraries were diluted to 2 nM per the Tapestation D1000 HS reading, pooled, and loaded on a NextSeq 500 for paired-end sequencing the following custom conditions: read 1: 66 cycles (no custom primer); index 1: 10 cycles (primers spiked in: oJBL432, oJBL494); read 2: 76 cycles (primers spiked in: oJBL433, oJBL334). oBC libraries were resequenced to improve saturation of the highly complex oBC libraries following: read 1: 34 cycles (no custom primers); index 1: 10 cycles (primer oJBL432); read 2 38 cycles (primer oJBL433). CS2 mBC libraries were sequenced separately, with: read 1: 30 cycles (no custom primer); index 1: 15 cycles, primer oJBL534; read 2: 18 cycles, primer oJBL334. For mBC and oBC libraries, read 1 provided the cell barcode and UMI, and read 2 the reporter barcode (sequenced with custom primers).