title: GSM8808514: HoLEP21 [HoLEP_21A_S82]; Homo sapiens; RNA-Seq
library_construction_protocol: Patients were positioned in the lithotomy position. The 2 lateral lobes and median lobe of the prostate were enucleated using a Holmium laser. Each lobe was dissected away from the surgical capsule in a distal to proximal approach and released from the bladder neck. Prostate tissue was minced and evacuated by a Piranha morcellator (Richard Wolf). Morcellation was performed according to manufacturer's recommendations up to 1,500 rpm with dual inflow saline irrigation. Morcellated tissue was collected in a canister and placed on ice prior to dissociation and preparation for single cell RNA-sequencing. Morcellated tissue was immediately delivered to the laboratory and further minced to ~1-5mm3 pieces with surgical scissors in a petri dish with 10 mL RP-10 (RPMI + 10% FBS) on ice for up to 5 mins and washed with cold RP-10 (RPMI + 10% FBS). Each sample was centrifuged at 1200 rpm x5 mins at 4°C and resuspended in 10 mL warmed digestive media (HBSS + 1% HEPES) with 1,000 U/mL collagenase type IV (Worthington, Cat: LS004188), and rotated for 30 min at 37 °C. Samples were triturated by pipetting 10 times at the end of the incubation. Each sample was filtered through a 70-µm filter (Falcon, Cat: 352350), washed with RP-10, centrifuged at 1200 rpm x5 mins at 4°C, washed again with RP-10, and resuspended in RP-10. Sequencing was largely based on the Seq-Well S^3 protocol. One to two arrays were used per sample. Each array was loaded as previously described with approximately 110,000 barcoded mRNA capture beads (ChemGenes, Cat: MACOSKO-2011-10(V+)) and with 15,000–20,000 cells. Arrays were sealed with functionalized polycarbonate membranes (Sterlitech, Cat: PCT00162X22100) and were incubated at 37 °C for 40 min. After sealing, each array was incubated in lysis buffer (5 M guanidine thiocyanate, 1 mM EDTA, 0.5% sarkosyl, 1% BME). After detachment and removal of the top slides, arrays were rotated at 50 rpm for 20 min. Each array was washed with hybridization buffer (2 M NaCl, 4% PEG8000) and was then rocked in a hybridization buffer for 40 min. Beads from different arrays were collected separately. Each array was washed ten times with wash buffer (2 M NaCl, 3 mM MgCl2, 20 mM Tris-HCl pH 8.0, 4% PEG8000) and scraped ten times with a glass slide to collect beads into a conical tube. For each array, beads were washed with Maxima RT buffer (ThermoFisher, Cat: EP0753) and resuspended in reverse transcription mastermix with Maxima RT buffer, PEG8000, Template Switch Oligo, dNTPs (NEB, Cat: N0447L), RNase inhibitor (Life Technologies, Cat: AM2696) and Maxima H Minus Reverse Transcriptase (ThermoFisher, Cat: EP0753) in water. Samples were rotated end-to-end, first at room temperature for 30 min and then at 52 °C overnight. Beads were washed once with TE-TW, once with TE-SDS, twice with TE-TW, and once with 10 mM Tris-HCl pH 8.0. They were treated with exonuclease I (NEB), rotating for 50 min at 37 °C. Beads were washed once with TE-SDS and twice with TE-TW. They were resuspended in 0.1 M NaOH and rotated for 5 min at room temperature. They were subsequently washed with TE-TW and TE. They were taken through second strand synthesis with Maxima RT buffer, PEG8000, dNTPs, dN-SMRT oligo, and Klenow Exo- (NEB, Cat: M0212L) in water. After rotating at 37 °C for 1 h, beads were washed twice with TE-TW, once with TE and once with water. KAPA HiFi Hotstart Readymix PCR Kit (Kapa Biosystems, Cat: KK2602) and SMART PCR Primer were used in whole transcriptome amplification (WTA). For each array, beads were distributed among 24 PCR reactions. Following WTA, three pools of eight reactions were made and were then purified using SPRI beads (Beckman Coulter), first at 0.6× and then at a 1× volumetric ratio. For each sample, one pool was run on an HSD5000 tape (Agilent, Cat: 5067–5592). The concentration of DNA for each of the three pools was measured via the Qubit dsDNA HS Assay kit (ThermoFisher, Cat: Q33230). Libraries were prepared for each pool, using 1000 pg of DNA and the Nextera XT DNA Library Preparation Kit. They were dual-indexed with N700 and N500 oligonucleotides.