Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: CROP-seq: Adherent cells were detached using Trypsin-EDTA (Gibco #25300-054), following standard cell culture practices. Cells were collected by centrifugation at 300 rcf for 5 min, washed once with PBS-0.01% BSA (freshly prepared on the day of the run), and resuspended in 1 ml of PBS-0.01% BSA. Cells were filtered through a 40 µm cell strainer to obtain a suspension of single-cells, which were counted using a CASY device. Single cells were then co-encapsulated with barcoded beads (ChemGenes #Macosko-2011-10) using an Aquapel-coated PDMS microfluidic device (FlowJEM), connected to syringe pumps (kdScientific) via polyethylene tubing with an inner diameter of 0.38 mm (Scicominc #BB31695-PE/2). Cells were supplied in PBS-0.01% BSA at a concentration of 220 cells/µl, barcoded beads were resuspended in Drop-seq lysis buffer at a concentration of 150 beads/µl. The flow rates for cells and beads were set to 1.6 ml/hour, while Droplet Generation Oil (BioRad #1864006) was run at 8 ml/hour. During the run, the barcoded bead solution was mixed by magnetic stirring with a mixing disk set to 1 jump/s. A typical run lasted between 35 and 40 min. In case of multiple runs per day, droplets were intermittently stored at 4 °C and processed together. Our most important modification to the protocol is an alternative way to break droplets, which recovers beads much more efficiently than in the original publication of Drop-seq (Macosko et al., 2015). After removing as much oil below the droplet layer as possible, we added 30 ml of 6x SSC buffer (Promega #V4261) and 1 ml of Perfluoroctanol (Sigma Aldrich #370533-25G) and shook the tube forcefully 6 times to break the droplets. Based on their large diameter, beads were then collected by syringe-filtering the solution through a 0.22 µm filter unit (Merck #SLGV033RS), washing 2x with 20 ml of 6x SSC buffer and eluting by turning the filter upside down and rinsing it with 3x 10 ml of 6x SSC buffer. Beads were then collected by centrifugation at 1,250 rcf for 2 min, setting the brake speed to 50%. After washing a second time with 10 ml 6x SSC, the pellet was taken up in 200 µl of 5x RT buffer and transferred to a 1.5 ml tube. Bulk RNA-seq: Total RNA was extracted using the Qiagen AllPrep Kit (Qiagen #80311). CROP-seq: Reverse transcription and Exonuclease I treatment were performed as described in the original publication, and the number of barcoded beads was estimated using a Fuchs-Rosenthal counting chamber (mixing the bead suspension with 6x DNA loading dye). Depending on the performance of the experiment, we prepared up to 24 PCR reactions per Drop-seq run, adding 4,400 beads (~220 cells) per PCR and enriching the cDNA for 4 + 10 cycles, using the already described reagents. We then prepared Drop-seq libraries using the Nextera XT kit (Illumina #15032350), starting from 1 ng of cDNA pooled in equal amounts from all PCR reactions for a given run. We typically required an additional 10 enrichment cycles, using the Illumina Nextera XT i7 primers along with the Drop-seq New-P5 SMART-PCR hybrid oligo. The slightly increased cDNA input typically results in an average size distribution of about 575 bp. After quality control, libraries were sequenced with paired-end SBS chemistry on Illumina HiSeq 3000/4000 instruments. Drop-seq Custom Read1 Primer was spiked into the HP10 primer solution, located in column 11 of the cBot Reagent Plate at 1µM final concentration. High sequence complexity needed for optimal base calling performance was achieved by adding 20-30% PhiX as spike-in. Cluster generation and Read 1 primer hybridization were completed using cBot protocol ‘HiSeq_3000_4000_HD_Exclusion_Amp_v1.0’. Alternatively, libraries were sequenced on an Illumina NextSeq 550 instrument using the 75 cycle High Output Kit. We loaded 1.8 pM library and provided Drop-seq Custom Read1 Primer at 0.3 µM in position 7 of the reagent cartridge. On NextSeq machines, we sequenced without PhiX spike-in, using a read configuration of 20 bases (Read1), 8 bases (Index) and 64 bases (Read2); Bulk RNA-seq: Bulk RNA-seq libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen #015.96) according to the manufacturer's instructions.