GSM4453990: S1b; Mus musculus; RNA-Seq
Single organoids collected by dissolving Matrigel with cold Cell Recovery Solution, followed by hand-picking and transferring to Nunc microwell culture plate with a dissociation mix containing protease isolated from B. licheniformis, EDTA, EGTA, DNase I, Accutase and PBS. Single organoids were dissociated by combining trituration using a siliconized pipette tips every 5 minutes and incubation at 37oC on for 15 minutes total. Following the dissociation, cell suspensions were diluted cell loading buffer in the loading tip of the DisCo chip. After bead-cell in droplet co-encapsulation, the gel loading tip containing the sample droplets was transferred to a bead collection chip inlet (cp-chip; as described in Lab on a chip). Droplets in the tip were flushed to bead collection chip. Subsequently to bead capture, washing was performed as in Drop-seq with SSC and Reverse transcription buffer directly in the recovery chip. Reverse transcription solution was added to the beads and the recovery chip was placed on a heating block to perform first strand cDNA synthesis (RT) for 90 minutes at 42oC. After RT reaction beads were washed on the recovery chip with TE-SDS once, with TE-TW twice and with Tris once. The beads were treated with Exonuclease I for 45 minutes at 37oC to remove single-stranded oligonucleotides on beads. After Exonuclease I treatment, beads were washed with TE-SDS once, with TE-TW twice (as after reverse transcription). Beads were then eluted from the recovery chip in dH2O. cDNA was amplified for 18 – 23 cycles using Kapa HiFi Hot start ready mix. cDNA was purified with CleanPCR magnetic beads using 0.6X ratio to remove small cDNA fragments and primers. To assess the cDNA yield and quality concentration was measured using Qubit and cDNA traces quality was assessed using Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (generated as described in Picelli et al..) and the library was purified using CleanPCR magnetic beads (0.6X ratio). Libraries were sequenced on a NextSeq 500 system (Illumina) following recommendations from original protocol (20 bp for read 1 and 50 bp for read2). 
GEO Accession
GSM4453990