title: GSM3103088: 74_NLW44_A_bulkRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Organoids collected from a single well were dissected in ice-cold PBS and finely minced in a petri dish on ice using razor blades. The tissue were added to 1 ml of cold active protease solution (PBS, 10 mg of Bacillus Licheniformis protease [Sigma, #P5380], 5 mM CaCl2, 20 U DNAse I [Roche, #4716728001]). The tissue was incubated in a 2 ml reaction tube for 15-20 min on a slow moving shaker (nutator) in a coldroom at 4°C with repeated trituration steps for 20 seconds every 5 minutes. Single cell dissociation was confirmed with a microscope. The dissociation was stopped with 1 ml ice cold PBS supplemented with 10% fetal bovine serum (FBS). Afterwards the cells were immediately pelleted at 300x g for 5 min at 4°C. Subsequently, the supernatant was discarded and cells were suspended in 2 ml PBS/10%FBS and pelleted again at 300x g for 5 min at 4°C. Then cells were suspended in PBS/0.01%BSA and pelleted again (300x g for 5 min at 4°C), suspended in 1 ml PBS/0.01%BSA, and passed through a 30 µM filter mesh (Miltenyi MACS smart strainer). Viability was then investigated with the Trypan-blue exclusion test and cell concentration was determined with a hemocytometer and adjusted to 200,000 cells/ml for Drop-seq. Uniformly dispersed 1 nl-sized droplets were generated using self-built polydimethylsiloxane (PDMS) microfluidic co-flow devices on the basis of the AutoCAD design provided by the McCarroll group. The devices were treated with a water repellant solution (Aquapel) to create a hydrophobic channel surface. Drop-Seq runs followed closely the procedure published by Macosko et al. (Online Dropseq protocol v. 3.1 http://mccarrolllab.com/dropseq/). Barcoded beads (ChemGenes Corp., Wilmington, MA), suspended in lysis buffer, were co-flown with a single cell suspension and a droplet generation mineral oil (QX200, Bio-Rad Laboratories). Resulting droplets were collected in a 50 ml tube and immediately disrupted after adding 30 ml high-salt saline-sodium citrate buffer (6xSSC) and 1 ml perfluorooctanol. Subsequently, captured mRNA's were reverse transcribed for 2 hours using 2,000 U of the Maxima H Minus Reverse Transcriptase (ThermoFisher) followed by an exonuclease treatment for 45 minutes to remove unextended primers. After two washing steps with 6xSSC buffer about 70,000 remaining beads (60% of input beads) were aliquoted (5,000 beads per 50 µl reaction) and PCR-amplified (5 cycles at 65˚C and 12 cycles at 67˚C annealing temperature). Aliquots of each PCR reaction were pooled and double-purified using 0.5x volume of Agencourt AMPure XP beads (# A63881, Beckman Coulter) and finally eluted in 10 µl EB buffer. Quality and quantity of the amplified cDNAs were analyzed on a BioAnalyzer High Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA). About 600 pg cDNA was fragmented and amplified (17 cycles) to generate a next-generation sequencing library by using the Nextera XT DNA sample preparation kit (Illumina). The libraries were purified, quantified (Agilent High sensitivity DNA chip), and then sequenced (paired end 26x115 bases) on the Illumina HiSeq2500 platform. Custom primer (5'-GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC-3') was used for the first sequence read to identify all different cell barcodes und unique molecular identifier (UMI) sequences.
attributes.tag: GEO Accession
attributes.value: GSM3103088