DSP (Lomant’s reagent; Thermo Fisher, cat. no. 22586 or PG82081)
CRITICAL DSP is sensitive to water and should be used immediately after dissolving in dimethyl sulfoxide (DMSO).
Methanol (Millipore Sigma, cat. no. 494437-2L)
DMSO (Millipore Sigma, cat. no. D2438-5X10ML)
CRITICAL DMSO used for dissolving DSP should be new and unopened so that water is not introduced. These smaller bottles are useful for this reason.
Sodium phosphate dibasic (Millipore Sigma, cat. no. S3264-250G)
Sodium phosphate monobasic monohydrate (Millipore Sigma, cat. no. 71507-250G)
Potassium phosphate monobasic (Millipore Sigma, cat. no. P9791-100G)
Sodium chloride (Millipore Sigma, cat. no. S3014-500G)
Potassium chloride (Millipore Sigma, cat. no. P9541-500G)
Magnesium chloride solution, 2 M (Millipore Sigma, cat. no. 68475-100ML-F)
Igepal CA-630 (Millipore Sigma, cat. no. I8896-50ML)
BSA, 20 mg/ml (New England Biolabs, cat. no. B9000S)
DEPC (Millipore Sigma, cat. no. D5758-25ML)
CAUTION Handle DEPC, and samples containing it, in a fume hood.
Sucrose (VWR, cat. no. 97061-428)
Triton X-100 (Millipore Sigma, cat. no. T8787-100ML)
Tween 20 (Thermo Fisher, cat. no. BP-337-100)
10× Dulbecco’s PBS (10× DPBS; Thermo Fisher, cat. no. 14200075)
Superscript IV reverse transcriptase (Thermo Fisher, cat. no. 18090200)
CRITICAL This protocol has not been tested with other reverse transcriptases.
T4 DNA Ligase (New England Biolabs, cat. no. M0202L)
CRITICAL The previous version of this protocol used Quick Ligase, but the buffer that is included with that enzyme interferes with the pelleting of the nuclei during centrifugation.
Tagmentase (Tn5 transposase), unloaded (Diagenode, cat. no. C01070010-20)
CRITICAL The amount of tagmentase added to Step 57 has been determined for this brand only.
Tn5-N7 oligo (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′, high-purity salt-free; Eurofins)
Mosaic End (ME) oligo (5′-/5Phos/CTGTCTCTTATACACATCT-3′, high-purity salt-free; Eurofins)
NEBNext mRNA second-strand synthesis module (New England Biolabs, cat. no. E6111L)
NEBNext high fidelity 2× PCR master mix (New England Biolabs, cat. no. M0541L)
dNTP mix (New England Biolabs, cat. no. N0447L)
Agencourt AMPure XP (Beckman Coulter, cat. no. A63882)
Yoyo dye (Thermo Fisher, cat. no. Y3601)
RNaseAlert kit (IDT, cat. no. 11-02-01-02)
RNaseZap (Thermo Fisher, cat. no. AM9780)
Elution buffer (EB, 10 mM Tris pH 8.5; Qiagen, cat. no. 19086)
Protease (Qiagen, cat. no. 19157)
CRITICAL Do not use any other protease/proteinase. This one can be heat-inactivated at the temperature and time listed in the protocol.
6% TBE Novex PAGE gels (10 wells; Thermo Fisher, cat. no. EC6262BOX)
UltraPure agarose (Thermo Fisher, cat. no. 16500-500)
Qubit double-stranded DNA high-sensitivity quantitation kit (Thermo, cat. no. Q32851)
sci-RNA-seq3 indexed primer plates at 10 μM dilution (standard desalting for purification; random bases do not need hand-mixing). The complete list of primers is found in Supplementary Table 1. The plates include the following: plate(s) of indexed oligo-dT RT primers (5′- /5Phos/CAGAGCNNNNNNNN[10bpRTindex]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′, where ‘N’ is any base; IDT), plate(s) of indexed ligation primers (5′- GCTCTG[9- or 10-bp ligation index]TACGACGCTCTTCCGATCT[reverse complement of ligation index]-3′), plate of indexed PCR P7 primers (5′-CAAGCAGAAGACGGCATACGAGAT[PCR P7 index]GTCTCGTGGGCTCGG-3′; IDT); and PCR P5 primers (this primer does not need to be indexed if you do only one plate of PCR) (5′-AATGATACGGCGACCACCGAGATCTACAC[PCR P5 index]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; IDT).