title: GSM3500951: E14_WT5; Mus musculus; RNA-Seq
library_construction_protocol: Drop-seq was performed largely as described (Macosko et al, Cell, 2015). Briefly, cortical cells were diluted to an estimated concentration of 400 cells/µL in Ca2+ and Mg2+ free HBSS with ion channel inhibitors and HEK293T cells were spiked in at a concentration of 3% of total cells (12 cells/µL) while barcoded beads (ChemGenes Corporation, catalogue number Macosko201110) were resuspended in lysis buffer to an estimated concentration of 400 beads/µL. Cells and beads were co-encapsulated with oil (QX200™ Droplet Generation Oil for EvaGreen, Biorad) using a microfluidics chip (Part number 3200455, Dolomite). Droplets of around 3 mL of aqueous volume (1.5 mL of cells and beads) were broken with perfluorooctanol in 30 mL of 6x SSC. The harvested beads were then washed twice with 6x SSC and hybridized RNA was reverse transcribed using Maxima H minus Reverse Transcriptase (ThermoFisher). Populations of 2,500 reverse-transcribed beads (~100 cells) were separately amplified with 13 cycles of PCR (primers, chemistry, and cycle conditions identical to those previously described) and PCR products were purified with 0.6x AMPure XP beads (Agencourt). cDNA from an estimated 12,000 E14.5 cells and 8,000 P0 cells were pooled, purified and tagmented with Nextera XT DNA Library Preparation kit (Illumina). Input cDNA (1 ng) from each replicate was amplified with custom primer P5_TSO_Hybrid and Nextera index primers (N701, N702, N703, N704, N711, N712, N715, N716 and N718). Tagmented samples were purified twice with 0.6x and 1x AMPure XP beads. All replicates were pooled and sequenced on one Illumina HiSeq 4000 flowcell (eight lanes) to avoid sequencing bias. Read 1 was 20 bp; bases 1-12 represent the cell barcode, bases 13-20 represent UMI. Read 2 was 50 bp and Read 3 (sample index) was 8 bp. The 8bp indexes are found within the Read 2 headers. Samples were de-multiplexed using bcl2fastq version 2.18.0.12.
attributes.tag: GEO Accession
attributes.value: GSM3500951